CLIC1 Cooperates with Integrins to Promote Thrombus Formation and Angiogenesis

Chloride intracellular channel 1 (CLIC1) is a member of a family of six highly homologous membrane proteins (CLIC1-6), which have been shown to be co-regulated with integrins suggesting their involvement in cell adhesion, migration and proliferation. CLIC1 is a metamorphic protein that functions as an oxidoreductase in the cytoplasm as well as an ion channel in the cell membrane. CLIC1 is upregulated in angiogenic endothelial and metastatic tumor cells. In addition, studies in CLIC1 knockout mice have shown that CLIC1 promotes platelet function. Here, we hypothesize that CLIC1 supports cell adhesive functions in platelets as well as endothelial cells and in doing so mediates critical vascular biological processes such as thrombus formation, vascular repair and angiogenesis.

To assess the role of CLIC1 in endothelial cells, we monitored human umbilical venous endothelial cells (HUVEC) after transfection with CLIC1 Smartpool® or non-targeted siRNA by phase contrast microscopy. This experiment revealed a significant anti-proliferative effect in CLIC1 knockdown cells in conventional 2-dimensional culture that coincided with reduced cell spreading. The deficit in spreading was reiterated after embedding the CLIC1-depleted HUVEC in a 3-dimensional matrix of fibrin clot and this loss in anchorage led to a dramatic increase in cell death.

Immunocytochemistry and fluorescence microscopy of HUVEC 10 minutes after initial cell attachment showed strong CLIC1 expression in nascent lamellipodia, where it colocalized with F-actin. Recruitment of CLIC1 to the cell membrane was associated with lamellipodia formation and was lost at later stages of adhesion when HUVEC began to generate stress fibers. CLIC1 cell membrane expression was inhibited after knocking down integrin β3 or treating HUVEC with the small molecule CLIC1 inhibitor IAA94. Notably, inhibiting CLIC1 or integrin β3 caused a significant reduction of F-actin assembly in lamellipodia suggesting that both molecules cooperate during the initial phase of cell spreading.

Paralleling our experiments with HUVEC, we observed that CLIC1 co-localizes with F-actin in the leading edges as well as in the cortical ring of newly attached platelets. Moreover, using flow cytometry, we detected robust CLIC1 cell surface expression in platelets after treatment with ADP, which was enhanced after addition of the β3 integrin ligand fibrinogen and reduced when we blocked integrins with peptides representing the integrin recognition motif RGD. These data suggest a close coordination of CLIC1 membrane recruitment with the adhesive machinery in activated platelets. Moreover, inhibition of CLIC1 relocation to the platelet membrane with IAA94 hindered activation of integrin αIIbβ3, which resulted in impaired platelet aggregation.

Lastly, we tested whether CLIC1 inhibition has antithrombotic effects in vivo. To this end, we assessed vasoocclusion in a murine dorsal skinfold chamber model where blood flow was monitored by intravital fluorescence microscopy. Intraperitoneal injections of mice with the CLIC1 inhibitor IAA94 (20mg/kg) 19 hours and 1 hour before the photochemical induction of thrombus formation caused a significant delay in vaso-occlusion in response to CLIC1 inhibition compared to vehicle-treated controls indicating that CLIC1 inhibition has a significant anti-thrombotic effects in vivo. Together, our data show that CLIC1 cooperates with integrins during cell adhesion and as such mediates functions related to platelet homeostasis, thrombus formation, and angiogenesis.