Fig. 2.

FVIII expression induces ER stress, oxidative stress, and apoptosis upon in vivo expression in liver. DNA expression vectors were delivered by tail-vein injection into WT C57BL/6 mice. After 24 h, blood and liver tissues were isolated for analysis. (A) Liver tissue sections were analyzed for immunolocalization of FVIII antigen. (B) FVIII antigen in plasma samples and liver extracts was measured by ELISA. FVIII activity in plasma samples was measured using the COAMATIC assay kit. For activity measurements, the background of murine FVIII activity was subtracted (0.35 U/ml). (C) Western blot analysis of liver tissue for detection of BiP, phospho-eIF2α, and CHOP. Densitometry indicated that BiP was increased three fold and eIF2α-P was increased two fold in mice injected with wtFVIII and BDD compared with 226/N6. Spliced Xbp1 mRNA in liver tissue was measured by real-time RT-PCR. (D) Fresh frozen liver sections were prepared and stained with 2 μM dihydroethidine hydrochloride for 30 min at 37 °C. Sections were analyzed by fluorescence microscopy. (E) Malondialdehyde was measured in liver homogenates. (F) Liver lysates were analyzed for GSH and GSH and oxidized glutathione content. Data represent the mean and SD from three different animals in B, E, and F.