In Vitro Protection of Human Endothelial Cells from Adenovirus Vector Toxicity by Activated Protein C.

High dose adenovirus vector administration in vivo has been associated with toxicity toward many cell types, including endothelial cells. Some of the prominent pathological features of an adenovirus vector death in a gene therapy trial included capillary leak syndrome and disseminated intravascular coagulation (DIC). We investigated the hypothesis that activated protein C (APC) might have a protective effect on primary human microvascular endothelial cells exposed to a first-generation adenovirus vector. We exposed primary human endothelial cells to a first-generation (E1, E3 deleted) adenovirus vector, AVC3FIX5 at concentrations ranging from 103 to 105 vector particles per cell and showed dose-dependent cell death as early as 6 hours (40% cell death at the highest dose). Phase contrast and immunofluorescence microscopy revealed that some cells died rapidly by primary necrosis while others died by apoptosis over a longer time course. By 40 hours, only 40% of the cells were viable. We then tested the effect of pretreatment of endothelial cells with APC concentrations ranging from 1 nM to 100 nM. Dose-dependent protection was seen in which cell death was reduced to 9 and 2 % at APC concentrations of 50 and 100 nM, respectively. We also tested the effect of timing of the APC treatment and showed that 1 hour pre-treatment or concurrent APC treatment were protective, but APC administered one hour after the adenovirus exposure was substantially less protective. This suggested that APC exerts its protective actions on endothelial cells either by interfering with early steps in the interaction of the vector with the cells, (e.g., vector entry) or by modulating death signaling pathways. It has been proposed that APC protects against cell damage in sepsis by interaction with the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1) on the endothelial cell surface to induce MCP-1 and other immunomodulatory genes by proteolytic signaling (Riewald et al., Science296:1880–1882, 2002). Other investigators have shown protective effects of APC for endothelial cells subjected to hypoxia through normalization of levels of p53, Bax, and Bcl-2 gene expression (Cheng et al., Nat Med9:338–342, 2003). The APC concentrations in our experiments that were maximally protective (50–100 nM) were of the same order of magnitude as was shown to be protective in vitro by these investigators. If APC can be shown to have a protective effect against adenovirus-induced endothelial cell toxicity and DIC in vivo, this may be a useful therapeutic strategy to explore as treatement of gene therapy vector toxicity. Cell Viability vs. APC Concentration Cell Viability vs. APC Concentration