Challenge with LPS or TNF-α Demonstrates Unique Differences in Primary Arterial Endothelial Cells Pretreated with Recombinant Human Activated Protein C.
Authors: Martin James A. , Joyce David E. , Balsara Rashna , Ploplis Victoria A. , Castellino Francis J. 
Published: October 2019

Abstract
A human recombinant form of the endogenous anticoagulant APC (rhAPC) has been approved for treatment of severe sepsis, a condition with 30-50% mortality and affecting 750,000 US patients per year. Clinical and in vitro studies show that rhAPC has pro-fibrinolytic, anti-inflammatory, and anti-apoptotic properties. In order to better understand the anti-inflammatory mechanism of rhAPC and its receptor EPCR on primary murine aortic endothelial cells (EC), responses were compared between wild type (WT) and low-expressing endothelial protein C receptor (EPCRδ/δ) EC by total RNA for specified endothelial inflammatory markers. The purpose was to determine the effect of rhAPC and low expression of EPCR on murine arterial EC responses to tumor necrosis factor alpha (TNF-α) or endotoxin (LPS). EC from C57BL/6 mice aorta, WT or EPCRδ/δ, were isolated, cultured, and positively selected for EC markers (CD105, CD106). EC in serum free media were pretreated with 5ug/mL rhAPC (Eli Lilly) for 16 hours followed by challenge with 100ng/mL TNF-α or 10ug/mL LPS for 8 hours. Total RNA was analyzed by Quantitative Real-time PCR (QRT-PCR) for CXC chemokines MIP-2 and KC, adhesion markers E-Selectin or ICAM-1, cytokines MCP-1 and IL-6, and NFκB-1. Mean +/− standard error of the mean for the time points (T0, 0.5hr, 1hr, 2hr, 4hr, and 8hr) after TNF-α or LPS were compared between treatment groups. Both TNF-α and LPS produced expected characteristic fold changes of RNA expression over the eight hour time period in the murine EC. Without rhAPC EPCRδ/δ EC showed a similar response compared to WT EC. When pretreated with rhAPC for 16 hours followed by LPS challenge, EC RNA transcript levels for CXC chemokines and adhesion markers were suppressed more in EPCRδ/δ compared to WT EC. When pretreated with rhAPC for 16 hours followed by TNF-α challenge, RNA transcript levels for CXC chemokines and adhesion markers were elevated or showed little change in WT EC and EPCRδ/δ EC compared to EC not given rhAPC. Nuclear factor NFκB-1 RNA was suppressed in both WT EC and EPCRδ/δ EC with rhAPC pretreatment and subsequent inflammatory agent (LPS or TNF-α). Most striking was the unexpected suppressed response of rhAPC pretreated EPCRδ/δ EC compared to WT EC after addition of either inflammatory agent. Further studies suggested that surface EPCR protein did not appear to be enhanced with any treatment combination, or with rhAPC alone. These results are consistent with previously reported endothelial cell specific rhAPC response of CXC chemokines and the ability of rhAPC to suppress other TNF-α mediated inflammatory responses (eg. MCP-1 and NFkB-1). In addition, rhAPC pretreatment appeared to suppress LPS mediated inflammatory responses, including CXC chemokines. The enhanced suppression of inflammatory responses seen in arterial EPCRδ/δ EC compared to WT EC remains unexplained. Results from this study also indicate primary murine arterial endothelial cells treated with rhAPC respond differently to challenge with TNF-α versus LPS.