The bispecific antibody Emicizumab is increasingly used for hemophilia A-treatment. However, its specificity for human factors IX and X (FIX, FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here we describe a novel mouse model allowing to examine Emicizumab function. Briefly, FVIII-deficient mice receive Emicizumab intravenously 24h before performing a tail clip-bleeding model. A second infusion with human FIX and FX is administered 5 min before bleeding. This approach generates consistent levels of Emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg dose) and of both FIX and FX (85 and 101 U/dL respectively, after dosing 100 U/kg). Plasmas from these mice display FVIII-like activity in a diluted aPTT and in thrombin generation assays, similar to human samples containing Emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared to mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted a FVIII-like activity of Emicizumab that corresponds to a dose of 4.5 U FVIII/kg (i.e. 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), Emicizumab provided additive activity to allow a complete bleeding arrest. This model could be useful for further in vivo analysis of Emicizumab.
Recent studies have shown that in addition to being major constituents of the atheromatous core, solid cholesterol crystals (CCs) promote atherosclerotic lesion development and rupture by causing mechanical damage and exerting cytotoxic and pro-inflammatory effects. These findings suggest that targeting CCs might represent a therapeutic strategy for plaque stabilization. However, little is known about how cholesterol crystallization is initiated in human atherothrombotic disease. Here, we investigated these mechanisms. We performed a thorough immunohistological analysis of non-embedded, minimally processed human aortic tissues, combining polarized light and fluorescence microscopy. We found that CC formation was initiated during the fatty streak to fibroatheroma transition in tight association with the death of intralesional smooth muscle cells (SMCs). Cholesterol-loaded human SMCs were capable of producing CCs in vitro, a process that was enhanced by type I collagen and by inhibition of autophagy and cholesterol esterification. The fibrous transition, which was characterized by increased type I collagen expression, was associated with changes in the expression of autophagy and cholesterol flux-related genes, including a decrease in the autophagic adapter p62 and an increase in the cholesterol intracellular transporter Niemann-Pick C1. Collagen was identified as a potent inducer of these changes in SMCs. Collagen-induced changes in cholesterol metabolism and autophagy flux in smooth muscle foam cells at the fibrolipid transition likely contribute to initiate cholesterol crystallization in human atherosclerosis. Also, our data are in support of a protective role of autophagy against CC formation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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