Platelet IκB kinase-β deficiency increases mouse arterial neointima formation via delayed glycoprotein Ibα shedding

Abstract

Objective: On the luminal surface of injured arteries, platelet activation and leukocyte-platelet interactions are critical for the initiation and progression of arterial restenosis. The transcription factor nuclear factor-κB is a critical molecule in platelet activation. Here, we investigated the role of the platelet nuclear factor-κB pathway in forming arterial neointima after arterial injury.

Methods and results: We performed carotid artery wire injuries in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice with a platelet-specific deletion of IκB kinase-β (IKKβ) (IKKβ(fl/fl)/PF4(cre)/LDLR(-/-)) and in control mice (IKKβ(fl/fl)/LDLR(-/-)). The size of the arterial neointima was 61% larger in the IKKβ(fl/fl)/PF4(cre)/LDLR(-/-) mice compared with the littermate control IKKβ(fl/fl)/LDLR(-/-) mice. Compared with the control mice, the IKKβ(fl/fl)/PF4(cre)/LDLR(-/-) mice exhibited more leukocyte adhesion at the injured area. The extent of glycoprotein Ibα shedding after platelet activation was compromised in the IKKβ-deficient platelets. This effect was associated with a low level of the active form of A Disintegrin And Metalloproteinase 17, the key enzyme involved in mediating glycoprotein Ibα shedding in activated IKKβ-deficient platelets.

Conclusions: Platelet IKKβ deficiency increases the formation of injury-induced arterial neointima formation. Thus, nuclear factor-κB-related inhibitors should be carefully evaluated for use in patients after an arterial intervention.

Figure 1

IKKβ deficiency in platelets augments injury-induced arterial neointima formation in LDLR^–/– mice. A, Movat pentachrome staining of the arterial neointima 4 weeks after injury; the size of the neointima and media were measured (n = 12 for each group). B, Immunostaining (with anti-F4/80) of the infiltrating macrophages in the arterial neointima. Twelve cross sections of each mouse carotid artery were analyzed. The percent positive area was calculated by dividing the positive area by the measured lesion area. Twelve injured carotid arteries were included for each group.

Figure 2

IKKβ deficiency in platelets increases leukocyte interactions with the injured arteries. A to C, The carotid arteries collected at 1 hour after wire injury were stained for platelets (A) and neutrophils (B), and the carotid arteries collected 7 days after the wire injury were stained for macrophages (C) (n = 5). D, Leukocyte rolling and adhesion in the injured carotid arteries within the first 25 min after injury (n = 5). E, Leukocyte rolling and adhesion on the activated IKKβ^fl/fl or IKKβ^fl/fl/PF4^cre platelets in the microflow chambers (n = 5).

Figure 3

IKKβ deficiency in platelets decreases platelet activation, secretion and aggregation. The washed platelets from the IKKβ^fl/fl/PF4^cre and the IKKβ^fl/fl mice were stimulated with or without thrombin. A, Platelet P-selectin expression was analyzed by flow cytometry after stimulation with 0.1 U/ml thrombin for 0, 10, and 60 min at 22°C, and the relative expression of P-selectin (mean fluorescence intensity) was compared (n = 5). B, ATP release and platelet aggregation were examined in a Lumi-Aggregometer after the platelets were incubated with 0.025 U/ml thrombin. The data represent 5 independent experiments. C, Platelet aggregation and α-granule release (arrowhead) were evaluated by electron microscopy after incubation with 0.1 U/ml thrombin for 10 min at 22°C. The data represent 5 independent experiments.

Figure 4

IKKβ deficiency inhibits thrombin-induced GPIbα shedding in platelets. Platelets isolated from IKKβ^fl/fl/PF4^cre and IKKβ^fl/fl mice were stimulated with 0.1 U/ml thrombin. A, Representative flow cytometric histograms and the relative expression of GPIbα at different times after stimulation (n = 5). B, An immunoblot of platelet GPIbα at different times after stimulation (n = 5).

Figure 5

Blocking the GPIbα binding site on Mac-1 prevents the increase in leukocyte interactions with injured arteries in IKKβ^fl/fl/PF4^cre/LDLR^–/– mice. A to B, Immunostaining of platelets and neutrophils on carotid arteries collected 1 hour after wire injury (n = 5). *P < 0.05 vs. IKKβ^fl/fl/LDLR^–/– mice treated with control IgG; ^#P < 0.05 vs. IKKβ^fl/fl/PF4^cre/LDLR^–/– mice treated with control IgG. C, Leukocyte rolling and adhesion in the carotid arteries within the first 25 min after injury (n = 5). White squares: IKKβ^fl/fl/LDLR^–/– mice treated with control IgG; white circles: IKKβ^fl/fl/PF4^cre/LDLR^–/– mice treated with control IgG; black squares: IKKβ^fl/fl/LDLR^–/– mice treated with anti-M2 antibody; black circles: IKKβ^fl/fl/PF4^cre/LDLR^–/– mice treated with anti-M2 antibody. *P < 0.05 vs. IKKβ^fl/fl/LDLR^–/– mice treated with control IgG; ^#P < 0.05 vs. IKKβ^fl/fl/PF4^cre/LDLR^–/– mice treated with control IgG. D, Leukocyte rolling and adhesion on the activated IKKβ-deficient and control platelets in the micro-flow chambers within the first 5 minutes (n = 5). White squares: wild type mice were treated with control IgG, and their blood was passed through a micro-flow chamber coated with control platelets; white circles: wild type mice were treated with control IgG, and their blood was passed through a micro-flow chamber coated with IKKβ-deficient platelets; black squares: wild type mice were treated with anti-M2 antibody, and their blood was passed through a micro-flow chamber coated with control platelets; black circles: wild type mice were treated with anti-M2 antibody, and their blood was passed through a micro-flow chamber coated with IKKβ-deficient platelets. *P<0.05 vs. wild type mice were treated with the control IgG and blood was passed through a micro-flow chamber coated with the control platelets; ^#P<0.05 vs. wild type mice treated with control IgG and blood was passed through a micro-flow chamber coated with IKKβ-deficient platelets.

Figure 6

IKKβ deficiency suppresses ADAM17 maturation in platelets. Platelets from IKKβ^fl/fl/PF4^cre and IKKβ^fl/fl mice were stimulated with 0.1 U/ml thrombin. A, Western blot analysis showing the levels of the immature (or pro) and mature (or active) ADAM17 in the platelets (n = 5). B, Western blot analysis showing the level of phosphorylated p38 MAPK in the platelets (n = 5).