Alternatively Spliced Tissue Factor promotes plaque progression, inflammation and angiogenesis in experimental atherosclerosis

Abstract

Introduction: Alternatively Spliced Tissue Factor (asTF) is novel isoform of tissue factor with angiogenic activity mediated via HIF-1α signaling (Giannarelli, AHA 13). asTF is highly expressed in human complicated atherosclerotic plaques (Giannarelli, ACC 13); however, it is unknown whether asTF has a functional role in atherogenesis. Hypothesis: asTF promotes atherosclerotic plaque progression, inflammation and angiogenesis. Methods: ApoE-/- mice (8-weeks old; n=15) were fed a Western-type diet from 2 weeks before surgery continuing through the experiment. Immediately after transluminal wire injury of the left common carotid artery (LCCA), LCCA was incubated with lentivirus encoding asTF-GFP (asTF+ group; n=10) or GFP (asTF- control group; n=5). Four weeks after, blood and spleen were collected for flow cytometry analysis of neutrophils and monocytes. LCCA was removed and processed for H&E, Oil-Red O staining and immunostaining for macrophages (MOMA-2 and MAC-3), vascular smooth muscle cells (VSMC, α-actin), endothelial cells (CD31) and HIF-1α. Results: Neointimal thickness and plaque lipid accumulation were significantly greater in asTF+ vs asTF- mice (Fig 1, A-C). An increase in plaque macrophages, neovessels and HIF-1α was observed in asTF+ vs asTF- (Fig 1, D-F). Medial thickness and VSMC density were similar between groups. Increased circulating neutrophils and Ly6Chigh (classical/inflammatory) monocytes were observed in asTF+ vs asTF- mice (Fig 1, G,H). In contrast, circulating Ly6Clow (patrolling) monocytes were significantly reduced (Fig 1, I). Similar findings were observed in the spleen (Fig 1, J-L). Conclusions: Our results demonstrate that asTF expressed within atherosclerotic lesions promotes plaque progression towards a more advanced phenotype and is associated with systemic proinflammatory status. These data makes asTF an attractive marker of plaque vulnerability and a potential therapeutic target for plaque stabilization.