ENDOTHELIAL AUTOPHAGY REGULATES ARTERIAL THROMBUS FORMATION IN MICE

Abstract

Arterial thrombosis occurs after rupture of an atherosclerotic plaque, wherein endothelial cells (ECs) become activated and express procoagulant molecules. The role of ECs in thrombosis has not been fully elucidated. Recent evidence suggests that loss of autophagy, an intracellular catabolic process, decreases von Willebrand factor secretion in cultured ECs and in mice. Inasmuch as ATG7 is a key catalyst of autophagy, we explored the potential role of autophagy in thrombogenesis using EC-specific ATG7 knockout (EC-ATG7-/-) mice and their wild-type (WT) littermate controls. Thrombosis was evaluated using intravital microscopy in FeCl3-induced carotid and mesenteric, and laser-induced cremaster artery injury models. Circulating platelet counts were enumerated and platelet surface expression of P-selectin and β3-integrin (β3) determined by fluorescence-activated cell sorting. Time to carotid artery occlusion was significantly prolonged in EC-ATG7-/- mice compared with WT mice (15.5±2.0 and 31.0±4.0 min, respectively; N=10/group; p=0.003). Time to mesenteric artery occlusion was also significantly longer in EC-ATG7-/- mice compared with WT mice (20.0±1.4 and 26.6±1.8 min, respectively; N=10/group; p=0.008). Thrombi in laser injured cremaster arterioles were examined in 38 WT and 33 EC-ATG7-/- mice. The time to maximum platelet fluorescence intensity in EC-ATG7-/- mice was significantly prolonged relative to that for WT mice (1.0±0.6 and 2.3±1.2 min, respectively; p<0.0001). Thrombi size, as determined by platelet fluorescence intensity, were significantly smaller in EC-ATG7-/- vs. WT mice, (1.8±0.7 and 8.2±2.9, respectively; x109; p=0.049). Despite these differences, platelet counts for WT and EC-ATG7-/- mice were not appreciably different (768±86 and 715±125, respectively; x106; N=10; p=0.3). In WT and EC-ATG7-/- mice, surface expression of β3 (68.4±24.4 and 42.8±21.1 AU, respectively; p=0.7) and P-selectin (6.0±1.2 and 5.2±0.8 AU, respectively; p=0.9) were also similar. Although EC-ATG7-/- mice exhibited extended occlusion times and reduced thrombi formation across all three models of thrombosis, platelet count, P-selectin levels and β3 expression were similar to those in WT mice. Taken together, these findings are evidence that endothelial autophagy contributes to thrombosis in a platelet-independent manner.