The Blood Monocyte - TNF - Endothelial Axis in Activation of Endothelial Tissue Factor in the Transgenic Sickle Mouse: Possible Relevance of Etanercept to Sickle Coagulopathy
Authors: Solovey Anna , Chang Liming Milbauer , Abdulla Fuad , Kollander Rahn , Marker Paul H , Kelm Robert , Vercellotti Gregory M , Hebbel Robert P 
Published: October 2019

Abstract
Abstract 1048
Peripheral blood monocytes can be pathogenic participants in vascular disease, and they are abnormally activated in sickle patients. As shown earlier, sickle mice have abnormally increased expression of endothelial tissue factor (eTF) in pulmonary veins (PV), expressed as % of PV positive for eTF. For HbS-BERK (eTF 36%, vs 9% for HbA-BERK controls) and S+SAntilles (eTF 28%, vs 7% for C57BL6 controls), abnormal eTF expression is chronic. For NY1DD, however, eTF switches from 7% at ambient air to 23% after exposure to hypoxia/reoxygenation (post-H/R). Having now examined roles of transcription factors Egr-1 and NFkB(p50), we find that H/R triggered expression of eTF in post-H/R NY1DD is: [a] dependent upon both Egr-1 and NFkB(p50) within peripheral blood mononuclear cells (PBMC), and dependent upon Egr-1 butnot NFkB(p50) in vessel wall endothelium. To examine the roles of PBMC and their product TNF in activating eTF expression we used three strategies. [A] First, transfusion (Tx) of PBMC from donor mice (DM) induced eTF in recipient mice (RM) as follows. At-air NY1DD RM exhibited eTF of 7% from Tx of at-air NY1DD DM PBMC, and eTF of 19% from Tx of post-H/R NY1DD DM PBMC. C57BL6 RM exhibited eTF of 10% from Tx of C57BL6 DM PBMC, 31% from Tx of S+SAntilles DM PBMC, and only 15% from Tx of PBMC from S+SAntilles DM also having NFkB(p50)−/−. HbA-BERK RM exhibited eTF of 9% from Tx of HbA-BERK DM PBMC, and 36% from Tx of HbS-BERK DM PBMC. [B] Second, we examined effect of TNF+LT blocker etanercept on PBMC in transfusion experiments. As expected, HbA-BERK RM exhibited eTF 36% from Tx of HbS-BERK PBMC obtained from etanercept-treated (3 mg/kg × 2) DM, and 38% from Tx of HbS-BERK PBMC obtained from DM treated with inactive control TNF-R-scrambled-peptide. On the other hand, HbA-BERK RM pre-treated with etanercept (same dose) vs control TNF-R-scrambled-peptide exhibited eTF of 11% and 33%, respectively from Tx of HbS-BERK DM PBMC. [C] Third, direct TNF blocking pre-treatment of NY1DD mice prevented the eTF increase from subsequent H/R (to 23% for the no pre-treatment controls): eTF of 5% (P=.000013) for pre-treatment with high-etanercept (10 mg/kg × 2), 12% (P=.00059) for pre-treatment with low-etanercept (3 mg/kg × 2), 19% (P=.053) for pre-treatment with IL1-blocker anakinra (10 mg/kg ×2), and 7% (P=.0016) for pretreatment withTNF-specific blocker infliximab (10 mg/kg ×2). For HbS-BERK mice having pre-existing eTF of 36%, extended treatment with etanercept (3 mg/kg, twice weekly × 3) reduced eTF to 27% (P=.026) while those receiving scrambled-peptide remained at 34%. Higher or longer etanercept may be more effective. Other murine treatments have shown us reversal of pre-existing eTF requires extended treatment. Since regulation of TF expression in endothelial cells and blood monocytes is similar, it is likely that these eTF effects would be paralleled by similar effects on monocyte TF expression. Thus, these results suggest that TNF blocking strategies could be helpful in mitigating effects of coagulation activation in the sickle context.

Disclosures:
No relevant conflicts of interest to declare.