CLIC1 supports mechanisms related to thrombosis and vascular repair
L. M. Knowles, P. Niewald, E. Ampofo, A. Drawz, I. Müller, H. Eichler, J. Pilch (Homburg, Germany)
Time: 11:00 - 12:00
Objective: Chloride intracellular channel 1 (CLIC1) has been shown to be involved in thrombus formation and angiogenesis but the functional context of CLIC1 action remains largely unexplored. The objective of this study is to determine if CLIC1 supports cell adhesive processes that are relevant for endothelial and platelet function.
Methods: Human umbilical venous endothelial cells (HUVEC) were treated with CLIC1 siRNA or the CLIC1 small molecule inhibitor IAA94 and probed for cell proliferation on plastic (2D) as well as cell invasion/survival after embedding in fibrin (3D). Platelets were treated with IAA94 to assess the effect of CLIC1 on aggregation. Flow cytometry was used to determine integrin alphaIIbbeta3 activation as well as CLIC1 cell surface expression on platelets. The subcellular localization of CLIC1 in HUVEC/platelets was analyzed with fluorescence or confocal microscopy. The effect of CLIC1 on thrombus formation in vivo was assessed by intravital fluorescence microscopy in a mouse dorsal skin fold chamber model.
Results: Treatment of HUVEC with CLIC1 siRNA or IAA94 had a strong anti-proliferative effect in 2D and caused significant reduction of invasion and survival in 3D. At the same time, we detected relocation of CLIC1 into lamellipodia (2D) and invadopodia (3D) in untreated HUVEC. This particular distribution of CLIC1 is functionally significant as we found reduced membrane ruffling in combination with increased stress fiber formation in CLIC1 siRNA-treated HUVEC. Relocation of CLIC1 into the cell periphery was also detectable in platelets, which expressed CLIC1 on the cell surface in an RGD-dependent manner. CLIC1 relocation to the platelet membrane was inhibited after treatment with IAA94, which also reduced integrin alphaIIbbeta3 activation. This in turn led to impaired platelet aggregation in vitro and prolonged vaso-occlusion in a mouse model of photo-chemical thrombus formation in vivo.
Conclusion: Our results show that CLIC1 is regulated by adhesive interactions with integrin ligands that cause CLIC1 to relocate to the cell surface. This process in turn appears to be relevant for integrin-mediated functions involved in platelet thrombus formation, angiogenesis and vascular repair.