Platelet IκB Kinase-β Deficiency Increases Mouse Arterial Neointima Formation via Delayed Glycoprotein Ib α Shedding Shujian Wei, Huan Wang, Guoying Zhang, Ying Lu, Xiaofei An, Shumei Ren, Yunmei Wang, Yuguo Chen, James G. White, Chunxiang Zhang, Daniel I. Simon, Chaodong Wu, Zhenyu Li and Yuqing Huo

Arterioscler Thromb Vasc Biol. 2013;33:241-248; originally published online December 13, 2012; doi: 10.1161/ATVBAHA.112.300781 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2012 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/33/2/241

Data Supplement (unedited) at: http://atvb.ahajournals.org/content/suppl/2012/12/13/ATVBAHA.112.300781.DC1.html

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document.

Reprints: Information about reprints can be found online at: http://www.lww.com/reprints

Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: http://atvb.ahajournals.org//subscriptions/

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 Platelet IκB Kinase-β Deficiency Increases Mouse Arterial Neointima Formation via Delayed Glycoprotein Ibα Shedding

Shujian Wei,* Huan Wang,* Guoying Zhang, Ying Lu, Xiaofei An, Shumei Ren, Yunmei Wang, Yuguo Chen, James G. White, Chunxiang Zhang, Daniel I. Simon, Chaodong Wu, Zhenyu Li, Yuqing Huo

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 κI B kinase-β (IKKβ) (IKKβfl/fl/PF4cre/LDLR–/–) and in control mice (IKKβfl/ fl/LDLR–/–). The size of the arterial neointima was 61% larger in the IKKβfl/fl/PF4cre/LDLR–/– mice compared with the littermate control IKKβfl/fl/LDLR–/– mice. Compared with the control mice, the IKKβfl/fl/PF4cre/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. Conclusion—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. (Arterioscler Thromb Vasc Biol. 2013;33:241-248.) Key Words: arterial injury ◼ leukocytes ◼ NF-κB ◼ platelets ◼ restenosis

eointimal hyperplasia after percutaneous interventions, regulates the activation of NF-κB is based on the degradation Nsuch as balloon angioplasty or stenting, is the principal of IκB from the NF-κB complex, a process initiated by IκB cause of arterial restenosis.1 Platelet deposition and subsequent phosphorylation. The latter is catalyzed by a multisubunit leukocyte–platelet interactions on the injured luminal surface protein kinase called IκB kinase (IKK).8 IKKβ is more active are critical in the repair process after arterial damage.2–5 than other IKK subunits in catalyzing IκB phosphorylation.9 Although platelets are anucleate cells, they contain nearly all The loss of IKKβ dramatically inhibits NF-κB activation, of the nuclear factor-κB (NF-κB) family members.6 NF-κB resulting in embryonic lethality in mice.10 plays a complex role in platelet activation. NF-κB inhibitors We were interested in determining whether the inhibition of impair platelet aggregation mediated by ADP, collagen, platelet NF-κB activation could suppress neointima formation and thrombin, and reduce ATP release, thromboxane B2 after arterial injury. Using floxed IKKβ (IKKβfl/fl) mice, we formation, and P-selectin expression stimulated by thrombin.7 generated mice with IKKβ deficiency specifically in platelets However, arachidonic acid-induced platelet activation is not by breeding PF4-Cre (PF4cre) mice with these floxed mice, affected by NF-κB inhibitors.7 The common pathway that and breeding the resulting mice with low-density lipoprotein

Received on: August 6, 2012; final version accepted on: November 19, 2012. From The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Department of Emergency, Qilu Hospital, Shandong University, Jinan, Shandong, China (S.W., Y.C.); Vascular Biology Center, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA (S.W., Y.L., X.A., Y.H.); Department of Medicine, University of Minnesota, Minneapolis, MN (H.W., J.G.W.); Division of Cardiovascular Medicine, the Gill Heart Institute, and Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY (G.Z., Z.L.); Department of Endocrinology, Jiangsu Province Hospital of Chinese Medicine, Nanjing, China (X.A.); Drug Discovery Center, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, China (S.R.); Harrington Heart & February Vascular Institute, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, OH (Y.W., D.I.S.); Department of Pharmacology, Rush Medical College, Rush University, Chicago, IL (C.Z.); and Department of Nutrition and Food Science, Texas A&M University, College Station, TX (C.W.). *These authors contributed equally to this work. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300781/-/DC1. Correspondence to Yuqing Huo, MD, PhD, Chief of Vascular Inflammation Program, Vascular Biology Center Department of Cellular Biology and Anatomy, Medical College of Georgia Georgia Health Sciences University Sanders Building, CB-3919A 1459 Laney Walker Blvd, Augusta, GA. E-mail 35,123,130,134,181 [email protected] © 2012 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.112.300781 35,123,181,123 Downloaded from http://atvb.ahajournals.org/ at Georgia Regents241 University Greenblatt Library on December 12, 2013 242 Arterioscler Thromb Vasc Biol February 2013 receptor-deficient (LDLR–/–) mice. The role of platelet IKKβ in arterial neointima formation was evaluated by comparing the size of the neointima and leukocyte–platelet interactions in the injured arteries. Furthermore, the underlying mechanisms contributing to the change in the leukocyte–platelet interac- tions were explored.

Materials and Methods The PF4cre mice11 and the floxed-IKKβ mice,12 provided by the groups of Drs Radek C. Skoda and Michael Karin, were bred. A mouse 384 single-nucleotide polymorphism panel (markers spread across the genome at approximately 7-Mbp intervals; Charles River Laboratories International, Inc, Wilmington, MA) was used to char- acterize the genetic background of the breeders. Polymorphic markers demonstrated that the IKKβfl/flPF4cre breeders were 98.17% C57BL/6. The LDLR–/– mice (002207) from the Jackson Laboratory were bred with IKKβfl/flPF4cre to generate IKKβfl/flPF4creLDLR–/– mice, and their littermate IKKβfl/flLDLR–/– mice. The breeding scheme is detailed in Figure I in the online-only Data Supplement. The mice were subjected to a guide wire injury in the carotid artery, and the size of the arterial neointima was examined using a method previously described.3,13,14 Leukocyte interactions with the injured arteries were studied by immunostaining the arteries with specific leukocyte markers or directly observing the injured arteries in vivo by intravital epifluorescence microscopy.3 All of the animal experiments and care were approved by the Georgia Health Sciences University Animal Care and Use Committee, in accordance with The Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. The data are presented as the mean±SEM and were analyzed by either 1-way ANOVA followed by a Bonferroni correction post hoc test or Student t test to evaluate 2-tailed levels of significance. The null hypothesis was rejected at P<0.05. Figure 1. IκB kinase-β (IKKβ) deficiency in platelets augments Results injury-induced arterial neointima formation in low-density lipo- protein receptor–deficient (LDLR–/–) mice. A, Movat pentachrome Platelet IKKβ Deficiency Increases staining of the arterial neointima 4 weeks after injury; the size of Neointimal Formation in LDLR–/– mice the neointima and media were measured (n=12 for each group). B, fl/fl cre –/– Immunostaining (with anti-F4/80) of the infiltrating macrophages IKKβ /PF4 /LDLR mice and their littermate control in the arterial neointima. Twelve cross sections of each mouse IKKβfl/fl/LDLR–/– mice were fed a Western diet for 2 weeks, carotid artery were analyzed. The percentage positive area was followed by carotid artery wire injury. Four weeks later, the calculated by dividing the positive area by the measured lesion arteries were excised and processed for analysis. The 2 groups area. Twelve injured carotid arteries were included for each group. of mice were identical in body weight, blood cholesterol lev- els, and number of peripheral leukocytes (Tables I and II in the control mice that retained IKKβ in their platelets. The carotid online-only Data Supplement). The number of platelets in the arteries of both groups were injured with a guide wire. Four IKKβfl/fl/PF4cre/LDLR–/– mice exhibited a decreasing trend, but weeks after the injury, we observed that the neointimal lesions –/– this trend did not reach statistical significance when compared were 64% larger in the LDLR mice that received bone fl/fl cre –/– with that in IKKβfl/fl/LDLR–/– mice (Table I in the online-only marrow from the IKKβ /PF4 /LDLR mice compared with Data Supplement). Surprisingly, the size of the neointima the control mice. The arterial neointimas from the LDLR–/– fl/fl cre –/– lesions was 61% larger in the IKKβfl/fl/PF4cre/LDLR–/– mice mice that received bone marrow from IKKβ /PF4 /LDLR compared with the IKKβfl/fl/LDLR–/– mice (Figure 1A). The mice also stained more strongly with a macrophage marker size of the media was also increased in the IKKβfl/fl/PF4cre/ (Figure IIIA and IIIB in the online-only Data Supplement). LDLR–/– mice compared with the IKKβfl/fl/LDLR–/– mice (Figure 1A). Notably, the ratio of the intima to the media Platelet IKKβ Deficiency Increases Leukocyte (I/M) was markedly elevated in the IKKβfl/fl/PF4cre/LDLR–/– Adhesion to Injured Carotid Arteries mice compared with the IKKβfl/fl/LDLR–/– mice (Figure II and Platelet–Leukocyte Aggregates in the online-only Data Supplement). Macrophages in the To evaluate the leukocyte interactions with the injured arteries, injured carotid arteries were stained with F4/80 and found to arterial cross sections were immunostained with specific mark- be significantly increased in the IKKβfl/fl/PF4cre/LDLR–/– mice ers for platelets, neutrophils, and monocytes. One hour after compared with the control mice (Figure 1B). the carotid arterial injury, platelets and leukocytes covered To exclude the effect of IKKβ deficiency in other PF4- the denuded luminal surface (Figure 2A and 2B), and almost expressing cells on the formation of arterial neointima,15 no monocytes were observed on the denuded luminal surface we used bone marrow transplantation to generate LDLR–/– (data not shown). The number of platelets covering the injured chimeric mice that lacked IKKβ only in their platelets and the area was not significantly different between the IKKβfl/fl/PF4cre/

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 Wei et al Platelets, IKKβ, and Arterial Injury 243

to the arterial walls of the IKKβfl/fl/PF4cre/LDLR–/– mice compared with the IKKβfl/fl/LDLR–/– mice (Figure 2D). To further define the binding affinity of the IKKβ-deficient and control platelets to leukocytes, an ex vivo microflow chamber was used.3,14 The chamber was coated with thrombin- activated, IKKβ-deficient and control platelets, which were isolated from IKKβfl/fl/PF4cre or IKKβfl/fl mice. Then, whole blood from the wild-type mice was allowed to flow through the chamber. Interestingly, the number of rolling wild-type leu- kocytes on the chamber coated with activated IKKβ-deficient platelets was low, but these levels did not reach significance when compared with the chamber coated with control platelets. However, the number of adhering leukocytes was much greater in the chamber coated with IKKβ-deficient platelets compared with the chamber coated with control platelets (Figure 2E). To evaluate the role of platelet IKKβ deficiency in medi- ating platelet–leukocyte interactions in the circulation, we examined the mouse blood leukocyte population by flow cytometry. Both platelet–neutrophil and platelet–monocyte aggregates were increased 4 to 5 times in the IKKβfl/fl/PF4cre/ LDLR–/– mice compared with the IKKβfl/fl/LDLR–/– mice (Figure IV in the online-only Data Supplement). Within those aggregates, the level of glycoprotein (GP) Ibα expres- sion was remarkably higher in IKKβfl/fl/PF4cre/LDLR–/– mice (Figure IV in the online-only Data Supplement). Leukocyte Mac-1 (αMβ2, CD11b/CD18) and platelet GPIbα are criti- cally involved in the formation of leukocyte–platelet aggre- gates.16–18 This result suggests that platelet IKKβ deficiency enhances platelet–leukocyte aggregation resulting from the elevated level of GPIbα on IKKβ-deficient platelets.

Platelet IKKβ Deficiency Decreases Platelet Activation, Secretion, and Aggregation Figure 2. IκB kinase-β (IKKβ) deficiency in platelets increases To evaluate the role of IKKβ deficiency in platelet func- leukocyte interactions with the injured arteries. A through C, The tion, platelets from IKKβfl/fl/PF4cre mice and their IKKβfl/fl carotid arteries collected at 1 hour after wire injury were stained for platelets (A) and neutrophils (B), and the carotid arteries col- littermates were stimulated with thrombin. Flow cytometry lected 7 days after the wire injury were stained for macrophages revealed that there was no significant difference in P-selectin (C; n=5). D, Leukocyte rolling and adhesion in the injured carotid expression between the resting IKKβ-deficient platelets and arteries within the first 25 minutes after injury (n=5).E , Leukocyte the control platelets (Figure 3A). However, P-selectin expres- rolling and adhesion on the activated IKKβfl/fl or IKKβfl/fl/PF4cre platelets in the microflow chambers (n=5). sion induced with 0.1 U/mL thrombin was diminished in the IKKβ-deficient platelets compared with the control platelets (Figure 3A). In addition, Lumi-Aggregometer–based experi- LDLR–/– and IKKβfl/fl/LDLR–/– mice (Figure 2A). However, ments demonstrated that IKKβ-deficient platelets exhibited considerably more neutrophils were adhered to the injured reduced ATP release and aggregation compared with control fl/fl cre –/– area of the carotid arteries in the IKKβ /PF4 /LDLR mice platelets, when stimulated with thrombin (Figure 3B). Electron fl/fl –/– compared with the IKKβ /LDLR mice (Figure 2B). Seven microscopy also indicated that the release of α-granules and days after the wire injury, more infiltrating macrophages were platelet aggregation were attenuated in the IKKβ-deficient fl/fl cre observed in the injured carotid arteries of the IKKβ /PF4 / platelets compared with the control platelets, after stimulation –/– fl/fl –/– LDLR mice than in the IKKβ /LDLR mice (Figure 2C). with thrombin for 10 minutes (Figure 3C). Next, we examined the interactions of the leukocytes with the injured mouse carotid arteries in vivo by using intravital IKKβ Deficiency Attenuates GPIbα epifluorescence microscopy. The circulating leukocytes, which Shedding in Platelets were labeled with rhodamine 6G, rolled on and adhered to the Platelet GPIbα plays an important role in the interaction injured vessel wall after the wire injury. The number of leukocytes between platelets and leukocytes.16–18 We examined the level rolling on the arterial wall did not differ between the IKKβfl/fl/ of GPIbα in IKKβ-deficient platelets. Platelets were iso- PF4cre/LDLR–/– and IKKβfl/fl/LDLR–/– mice (Figure 2D). During lated from IKKβfl/fl/PF4cre mice and their littermate IKKβfl/fl the early stage after the arterial injury, the number of leukocytes mice. Flow cytometry (Figure 4A) and Western blot analyses adhering to the arterial wall was not markedly different between (Figure 4B) demonstrated that there was no significant differ- the 2 groups. Twenty minutes later, more leukocytes had adhered ence in GPIbα expression between the resting IKKβ-deficient

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 244 Arterioscler Thromb Vasc Biol February 2013

Figure 3. IκB kinase-β (IKKβ) deficiency in plate- lets decreases platelet activation, secretion, and aggregation. The washed platelets from the IKKβfl/fl/ PF4cre and the IKKβfl/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 minutes 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 plate- lets 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 micros- copy after incubation with 0.1 U/mL thrombin for 10 minutes at 22°C. The data represent 5 independent experiments.

platelets and the control platelets. Stimulation with throm- was observed between the IKKβ-deficient and control plate- bin caused GP shedding from the surface of the platelets. lets (data not shown). No difference was observed in the pro- Indeed, thrombin treatment led to rapid GPIbα shedding from tein levels of GPV, GPVI, GPIX, or αIIbβ3 on the membrane the IKKβfl/fl platelets in a time-dependent manner. However, of the platelets after stimulation with ADP or collagen (data IKKβ deficiency significantly attenuated the extent of GPIbα not shown). These results demonstrate that platelet IKKβ shedding (Figure 4A and 4B). Flow cytometry demonstrated plays a critical role in GPIbα and GPV shedding after throm- slow shedding of GPV in IKKβ-deficient platelets compared bin stimulation. with control platelets (Figure VA in the online-only Data Supplement). Interestingly, stimulation with thrombin did not Blocking the GPIbα-Binding Site on Mac- change the surface levels of GPVI (Figure VB in the online- 1 Eliminates Increased Interactions Between only Data Supplement), GPIX (Figure VC in the online-only IKKβ-Deficient Platelets and Leukocytes Data Supplement), or αIIbβ3 (Figure VD in the online-only To further determine whether the increased leukocyte adhesion Data Supplement) on the platelets. ADP (5 μM) and collagen to the injured arteries was a result of the reduced GPIbα IV (20 μg/mL) also caused GPIbα shedding, but no difference shedding in IKKβfl/fl/PF4cre/LDLR–/– mice, we used an anti-M2

Figure 4. IκB kinase-β (IKKβ) deficiency inhibits thrombin-induced glycoprotein (GP) Ibα shedding in platelets. Platelets isolated from IKKβfl/fl/PF4cre and IKKβfl/fl mice were stimulated with 0.1 U/mL throm- bin. 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).

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 Wei et al Platelets, IKKβ, and Arterial Injury 245 antibody that selectively blocks GPIbα-binding site on Mac-1 in mouse injury models.18 The carotid arteries collected from mice at 1 hour after the carotid arterial injury were examined by histology. The number of platelets covering the injured area was not significantly different between the IKKβfl/fl/PF4cre/ LDLR–/– and the IKKβfl/fl/LDLR–/– mice treated with the control immunoglobulin G (IgG) or the anti-M2 antibody (Figure 5A). However, treatment with the anti-M2 antibody significantly reduced neutrophil adherence to the injured area of the carotid arteries in both the IKKβfl/fl/PF4cre/LDLR–/– mice and the IKKβfl/ fl/LDLR–/– mice compared with mice treated with control IgG (Figure 5B). More importantly, the increased leukocyte adhesion observed in the control IgG-treated IKKβfl/fl/PF4cre/ LDLR–/– mice compared with the control IgG-treated IKKβfl/ fl/LDLR–/– mice was abolished when these mice were treated with the anti-M2 antibody (Figure 5B). The same results were observed when we directly examined leukocyte interactions with the injured arteries by intravital epifluorescence microscopy (Figure 5C). In addition, in an ex vivo microflow chamber, the rolling of leukocytes on the activated IKKβ-deficient platelets or the control platelets did not differ significantly. In line with the in vivo data for leukocyte adhesion, the adhesion of the IgG-treated leukocytes to the activated IKKβ-deficient platelets in this ex vivo model was much greater than that observed in the activated control platelets. However, treatment with the anti-M2 antibody eliminated the increased leukocyte adhesion on the activated IKKβ-deficient platelets (Figure 5D).

IKKβ Deficiency Suppresses ADAM17 Maturation in Platelets The sheddase A Disintegrin And Metalloproteinase 17 (ADAM17) is critically involved in platelet surface receptor shedding.19,20 To test the involvement of ADAM17 in our system, we stimulated IKKβ-deficient and control platelets with thrombin and detected immature (or pro) and mature Figure 5. Blocking the glycoprotein (GP) Ibα-binding site on Mac-1 prevents the increase in leukocyte interactions with injured (or active) forms of ADAM17 using Western blot analysis. arteries in IKKβfl/fl/PF4cre/LDLR–/– mice. A and B, Immunostaining of The proform of ADAM17 was present in the resting and platelets and neutrophils on carotid arteries collected 1 hour after activated platelets (Figure 6A). The active form of ADAM17 wire injury (n=5). *P<0.05 vs IKKβfl/fl/LDLR–/– mice treated with con- # fl/fl cre –/– was detectable in the resting control platelets and transiently trol immunoglobulin G (IgG); P<0.05 vs IKKβ /PF4 /LDLR mice treated with control IgG. C, Leukocyte rolling and adhesion in the increased by 2.7-fold during platelet activation. This finding carotid arteries within the first 25 minutes after injury (n=5). White is consistent with earlier studies.20,21 IKKβ-deficient platelets squares indicate IKKβfl/fl/LDLR–/– mice treated with control IgG; had a similar level of active ADAM17 in the resting platelets, white circles, IKKβfl/fl/PF4cre/LDLR–/– mice treated with control IgG; black squares, IKKβfl/fl/LDLR–/– mice treated with anti-M2 antibody; but a much lower level of active ADAM17 during platelet and black circles, IKKβfl/fl/PF4cre/LDLR–/– mice treated with anti-M2 activation compared with the control platelets (Figure 6A). antibody. * indicates P<0.05 vs IKKβfl/fl/LDLR–/– mice treated with The level of phosphorylated p38 mitogen-activated protein control IgG; and #, P<0.05 vs IKKβfl/fl/PF4cre/LDLR–/– mice treated kinase (MAPK) has been shown to play a critical role in with control IgG. D, Leukocyte rolling and adhesion on the acti- 22–24 vated IκB kinase-β (IKKβ)-deficient and control platelets in the ADAM17 activation. Thus, we tested the effect of IKKβ microflow chambers within the first 5 minutes (n=5). White squares deficiency on p38 MAPK phosphorylation. Thrombin indicate wild-type mice were treated with control IgG, and their remarkably increased p38 MAPK phosphorylation in the blood was passed through a microflow chamber coated with con- IKK fl/fl platelets, but no significant increase in p38 MAPK trol platelets; white circles, wild-type mice were treated with con- β trol IgG, and their blood was passed through a microflow chamber phosphorylation was observed in the IKKβ-deficient platelets coated with IKKβ-deficient platelets; black squares, wild-type mice after 30 minutes of thrombin stimulation (Figure 6B). These were treated with anti-M2 antibody, and their blood was passed data indicate that IKKβ is a key molecule in ADAM17 through a microflow chamber coated with control platelets; and black circles, wild-type mice were treated with anti-M2 antibody, maturation via the regulation of p38 MAPK phosphorylation. and their blood was passed through a microflow chamber coated with IKKβ-deficient platelets. *P<0.05 vs wild-type mice were Discussion treated with the control IgG, and blood was passed through a microflow chamber coated with the control platelets; and# P<0.05 The initiation and progression of the neointima formation vs wild-type mice treated with control IgG, and blood was passed that underlies restenosis involves the adhesive interactions of through a microflow chamber coated with IKKβ-deficient platelets.

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 246 Arterioscler Thromb Vasc Biol February 2013

rapidly activates the leukocyte integrin Mac-1.31–33 The binding of leukocyte Mac-1 to platelet GPIbα subsequently strengthens the firm adhesion and transmigration of leukocytes to sites of platelet deposition.16–18 The loss of Mac-1/GPIbα binding leads to reduced leukocyte accumulation after arterial injury, and further results in the inhibition of neointima thickening.18 In this study, increased neointima formation in the IKKβfl/fl/PF4cre/ LDLR–/– mice was accompanied by an increase in leukocyte adhesion to the injured area. This increased neointima was eliminated by neutrophil depletion (Figure VI in the online- only Data Supplement), indicating that this phenotypic change in neointima formation is because of increased leukocyte adhesion. Furthermore, this increased leukocyte adhesion was abrogated by a blocking antibody that specifically inhibits the binding of leukocyte Mac-1 with platelet GPIbα.18 These results demonstrate that high levels of GPIbα on activated platelets contribute to the increased neointima formation in IKKβfl/fl/ PF4cre/LDLR–/– mice. The dynamic change in GPIbα on activated platelets occurs mainly through ADAM17-mediated GPIbα shedding. ADAM17 is a sheddase on the cell surface that cleaves a vari- ety of substrates, such as heparin-binding epidermal growth factor, transforming growth factor-α, tumor necrosis factor receptor, epidermal growth factor receptor, vascular cell adhe- sion molecule-1, and L-selectin.34 After cellular activation, the proform (134 kDa) of ADAM17 is proteolyzed to yield the mature (active) form (98 kDa). The latter active form cleaves its substrates.35 ADAM-17 activation in leukocytes occurs through p38 or the extracellular signal-regulated kinases 36,37 Figure 6. IκB kinase-β (IKKβ) deficiency suppresses A Disinteg- MAPK pathway. ADAM17 is a major sheddase involved in rin And Metalloproteinase 17 (ADAM17) maturation in platelets. platelet GPIbα shedding.19 ADAM17 inactivation led to about Platelets from IKKβfl/fl/PF4cre and IKKβfl/fl mice were stimulated a 90% reduction in GPIbα shedding in platelets. Brill et al with 0.1 U/mL thrombin. A, Western blot analysis showing the levels of the immature (or pro) and mature (or active) ADAM17 reported that oxidative stress activates ADAM-17 in platelets in the platelets (n=5). B, Western blot analysis showing the level in a p38-dependent fashion.23 Through a similar mechanism, of phosphorylated p38 mitogen-activated protein kinase in the IKKβ deficiency inhibited ADAM-17 maturation, resulting in platelets (n=5). delayed shedding of platelet GPIbα. This result is consistent with the observation that, in oral squamous cell carcinoma platelets with leukocytes, and leukocytes with the denuded ves- cells, NF-κB inhibition suppresses ADAM-17 maturation.38 sel wall.4,5 NF-κB is expressed in platelets and plays a critical Platelet IKKβ deficiency did not cause a significant change role in platelet activation.6,7 Here, we showed, in vivo, that plate- in platelet adhesion and accumulation on the injured arteries. let IKKβ deficiency increases neointima formation after arterial In this study, we found that IKKβ deficiency delayed GPIbα injury as a result of enhanced leukocyte–platelet interactions. and GPV shedding. GPIbα and GPV are able to bind von Furthermore, we found that IKKβ deficiency inhibits ADAM17 Willebrand factor and collagen, respectively.39,40 High levels maturation, resulting in delayed GPIbα shedding in platelets. of GPIbα and GPV may enhance platelet adhesion to the sub- Wire injury–induced neointima formation in the mouse endothelial area of the injured arteries.39,40 However, many carotid artery is a widely used model to mimic the pathology of other molecules that are important for platelet adhesion to the arterial neointima formation in patients with arterial restenosis.25 subendothelial area, including GPVI, GPIX, and αIIbβ3, did An excessive leukocyte–platelet interaction and leukocyte not differ between the IKKβ-deficient and the control platelets infiltration after artery injury has been shown to exaggerate (Figure V in the online-only Data Supplement).40–42 In addi- the repair mechanisms and augment neointima formation.26,27 tion, studies from other groups using NF-κB inhibitors have After endothelial denudation by mechanical injury, platelets shown that broad NF-κB inhibition causes a defect in platelet immediately adhere to and accumulate on the injured luminal adhesion and spreading.6,7 All of these factors may explain surface of arteries.2,3 P-selectin and many GPs on activated why decreased shedding of GPIbα and GPV does not result in platelets mediate leukocyte rolling and localization, and further an increase in platelet adhesion on the injured arteries. support leukocyte recruitment.28,29 The binding of platelet NF-κB is a double-edged sword for activated platelet-medi- P-selectin to its leukocyte ligand P-selectin glycoprotein ligand-1 ated pathologies. NF-κB is a key regulator of inflammation, initiates leukocyte recruitment to the activated platelets.30 The immunity, apoptosis, and cell proliferation in all types of nucle- interaction of P-selectin and P-selectin glycoprotein ligand-1 ated cells.43 NF-κB affects the progression of inflammatory

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 Wei et al Platelets, IKKβ, and Arterial Injury 247 diseases, such as myocardial ischemia, bronchial asthma, deficiency protects injured arteries from neointima formation in ApoE- arthritis, and cancer.44 The suppression of NF-κB activation has deficient mice. Arterioscler Thromb Vasc Biol. 2009;29:1053–1059. 4. Schober A, Weber C. Mechanisms of monocyte recruitment in vascular been shown to inhibit the expression of adhesion molecules, repair after injury. Antioxid Redox Signal. 2005;7:1249–1257. and the release of chemokines and cytokines by various inflam- 5. Manka DR, Wiegman P, Din S, Sanders JM, Green SA, Gimple LW, matory cells, eventually suppressing the progression of inflam- Ragosta M, Powers ER, Ley K, Sarembock IJ. Arterial injury increases 45–47 expression of inflammatory adhesion molecules in the carotid arteries of mation. However, NF-κB activation during the late stage of apolipoprotein-E-deficient mice. J Vasc Res. 1999;36:372–378. inflammation is associated with the resolution of inflammation 6. Spinelli SL, Casey AE, Pollock SJ, Gertz JM, McMillan DH, Narasipura and antiinflammatory gene expression.48 A few reports have SD, Mody NA, King MR, Maggirwar SB, Francis CW, Taubman indicated that blockade of the NF-κB pathway accelerates the MB, Blumberg N, Phipps RP. Platelets and megakaryocytes contain functional nuclear factor-kappaB. Arterioscler Thromb Vasc Biol. pathology of inflammatory diseases. For example, Kanter et 2010;30:591–598. al49 revealed that the inhibition of NF-κB activation in mac- 7. Malaver E, Romaniuk MA, D’Atri LP, Pozner RG, Negrotto S, rophages increased atherosclerosis in LDLR–/– mice. Zaph Benzadón R, Schattner M. NF-kappaB inhibitors impair platelet activa- et al50 reported that deficient NF-κB activation in the intesti- tion responses. J Thromb Haemost. 2009;7:1333–1343. 8. Huxford T, Huang DB, Malek S, Ghosh G. The crystal structure of the nal epithelium is associated with increased inflammation in IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB vivo. Consistent with its effect on inflammation, NF-κB activa- inactivation. Cell. 1998;95:759–770. tion initiates platelet activation. This process is evident by the 9. Li J, Peet GW, Pullen SS, Schembri-King J, Warren TC, Marcu KB, Kehry MR, Barton R, Jakes S. Recombinant IkappaB kinases reduced level of P-selectin, and the suppressed release of gran- alpha and beta are direct kinases of Ikappa Balpha. J Biol Chem. ules and ATP in IKKβ-deficient platelets. However, NF-κB 1998;273:30736–30741. activation appears to be necessary for activated platelets to 10. Tanaka M, Fuentes ME, Yamaguchi K, Durnin MH, Dalrymple SA, shed their GPs, and the inactivation of NF-κB led to sustained Hardy KL, Goeddel DV. Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice. Immunity. activation of platelets, as demonstrated by much higher lev- 1999;10:421–429. els of GPIbα and GPV on the surface of the activated IKKβ- 11. Tiedt R, Schomber T, Hao-Shen H, Skoda RC. PF4-Cre transgenic deficient platelets compared with the wild-type platelets. mice allow the generation of lineage-restricted gene knockouts for NF- B has been shown to be involved in the transcriptional studying megakaryocyte and platelet function in vivo. Blood. 2007;109: κ 1503–1506. regulation of >150 genes, a significant proportion of which 12. Pasparakis M, Courtois G, Hafner M, Schmidt-Supprian M, Nenci exhibit proinflammatory properties.51 Therefore, approaches to A, Toksoy A, Krampert M, Goebeler M, Gillitzer R, Israel A, Krieg specifically inhibit the NF-κB pathway are under active devel- T, Rajewsky K, Haase I. TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature. 2002; opment as possible therapeutic interventions. In this study, we 417:861–866. demonstrated that platelet IKKβ deficiency enhances leuko- 13. An G, Wang H, Tang R, Yago T, McDaniel JM, McGee S, Huo Y, Xia L. cyte–platelet interactions, resulting in aggregated neointima P-selectin glycoprotein ligand-1 is highly expressed on Ly-6Chi mono- formation after arterial injury. The underlying mechanism is cytes and a major determinant for Ly-6Chi monocyte recruitment to sites of atherosclerosis in mice. Circulation. 2008;117:3227–3237. that NF-κB inactivation delays ADAM17-mediated GPIbα 14. Wang H, Zhang W, Tang R, Zhu C, Bucher C, Blazar BR, Geng JG, shedding, thus strengthening the interaction of leukocytes Zhang C, Linden J, Wu C, Huo Y. Adenosine receptor A2A deficiency with platelets. Because the inhibition of the NF-κB pathway is in leukocytes increases arterial neointima formation in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2010;30:915–922. likely to be a promising therapeutic strategy and because tissue- 15. Ravid K, Beeler DL, Rabin MS, Ruley HE, Rosenberg RD. Selective specific therapies are not currently available, the application of targeting of gene products with the megakaryocyte platelet factor 4 pro- NF-κB inhibitors for diseases that have pathologies in which moter. Proc Natl Acad Sci USA. 1991;88:1521–1525. activated platelets are extensive participants, such as arterial 16. Simon DI, Chen Z, Xu H, Li CQ, Dong J, McIntire LV, Ballantyne CM, Zhang L, Furman MI, Berndt MC, López JA. Platelet glycoprotein injury, sepsis, and thrombosis, should be carefully evaluated. ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/ CD18). J Exp Med. 2000;192:193–204. Sources of Funding 17. Ehlers R, Ustinov V, Chen Z, Zhang X, Rao R, Luscinskas FW, Lopez J, Plow E, Simon DI. Targeting platelet-leukocyte interactions: identifica- This work was supported by grants from the American Diabetes tion of the integrin Mac-1 binding site for the platelet counter receptor Association (1-10-BS-76 to Y.H., 1-10-JF-54 to C.W.), the American glycoprotein Ibalpha. J Exp Med. 2003;198:1077–1088. Heart Association (10GRNT4400005 to Y.H., 12BGIA9050003 to 18. Wang Y, Sakuma M, Chen Z, Ustinov V, Shi C, Croce K, Zago AC, C.W), the National Institutes of Health (HL080569, HL095556, and Lopez J, Andre P, Plow E, Simon DI. Leukocyte engagement of platelet HL108922 to Y.H., HL57506 MERIT Award to D.I.S.), the Joint glycoprotein Ibalpha via the integrin Mac-1 is critical for the biological Research Fund for Overseas Chinese Young Scholars (31028008, response to vascular injury. Circulation. 2005;112:2993–3000. to Y.H. and C.Z.), and the National Key Basic Research Program of 19. Bergmeier W, Piffath CL, Cheng G, Dole VS, Zhang Y, von Andrian China (2012CB910402 to Y.H.). UH, Wagner DD. Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo. Circ Res. 2004;95:677–683. Disclosures 20. Rabie T, Strehl A, Ludwig A, Nieswandt B. Evidence for a role of None. ADAM17 (TACE) in the regulation of platelet glycoprotein V. J Biol Chem. 2005;280:14462–14468. 21. Zhu L, Bergmeier W, Wu J, Jiang H, Stalker TJ, Cieslak M, Fan R, References Boumsell L, Kumanogoh A, Kikutani H, Tamagnone L, Wagner DD, 1. Davies MG, Hagen PO. Pathobiology of intimal hyperplasia. Br J Surg. Milla ME, Brass LF. Regulated surface expression and shedding support 1994;81:1254–1269. a dual role for semaphorin 4D in platelet responses to vascular injury. 2. Coller BS. Binding of abciximab to alpha V beta 3 and activated alpha Proc Natl Acad Sci USA. 2007;104:1621–1626. M beta 2 receptors: with a review of platelet-leukocyte interactions. 22. Canault M, Duerschmied D, Brill A, Stefanini L, Schatzberg D, Cifuni Thromb Haemost. 1999;82:326–336. SM, Bergmeier W, Wagner DD. p38 mitogen-activated protein kinase 3. Wang H, Zhang W, Tang R, Hebbel RP, Kowalska MA, Zhang C, Marth activation during platelet storage: consequences for platelet recovery and JD, Fukuda M, Zhu C, Huo Y. Core2 1-6-N-glucosaminyltransferase-I hemostatic function in vivo. Blood. 2010;115:1835–1842.

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 248 Arterioscler Thromb Vasc Biol February 2013

23. Brill A, Chauhan AK, Canault M, Walsh MT, Bergmeier W, Wagner DD. 38. Takamune Y, Ikebe T, Nagano O, Shinohara M. Involvement of Oxidative stress activates ADAM17/TACE and induces its target recep- NF-kappaB-mediated maturation of ADAM-17 in the invasion of tor shedding in platelets in a p38-dependent fashion. Cardiovasc Res. oral squamous cell carcinoma. Biochem Biophys Res Commun. 2009;84:137–144. 2008;365:393–398. 24. Duerschmied D, Canault M, Lievens D, Brill A, Cifuni SM, Bader 39. Moog S, Mangin P, Lenain N, Strassel C, Ravanat C, Schuhler S, Freund M, Wagner DD. Serotonin stimulates platelet receptor shedding by M, Santer M, Kahn M, Nieswandt B, Gachet C, Cazenave JP, Lanza tumor necrosis factor-alpha-converting enzyme (ADAM17). J Thromb F. Platelet glycoprotein V binds to collagen and participates in platelet Haemost. 2009;7:1163–1171. adhesion and aggregation. Blood. 2001;98:1038–1046. 25. Lindner V, Fingerle J, Reidy MA. Mouse model of arterial injury. Circ 40. Massberg S, Gawaz M, Grüner S, Schulte V, Konrad I, Zohlnhöfer Res. 1993;73:792–796. D, Heinzmann U, Nieswandt B. A crucial role of glycoprotein VI for 26. Libby P, Ganz P. Restenosis revisited–new targets, new therapies. N Engl platelet recruitment to the injured arterial wall in vivo. J Exp Med. J Med. 1997;337:418–419. 2003;197:41–49. 27. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 41. Grüner S, Prostredna M, Schulte V, Krieg T, Eckes B, Brakebusch C, 1999;340:115–126. Nieswandt B. Multiple integrin-ligand interactions synergize in shear- 28. Kuijper PH, Gallardo Torres HI, Houben LA, Lammers JW, Zwaginga resistant platelet adhesion at sites of arterial injury in vivo. Blood. JJ, Koenderman L. P-selectin and MAC-1 mediate monocyte rolling and 2003;102:4021–4027. adhesion to ECM-bound platelets under flow conditions. J Leukoc Biol. 42. Smyth SS, Reis ED, Zhang W, Fallon JT, Gordon RE, Coller BS. 1998;64:467–473. Beta(3)-integrin-deficient mice but not P-selectin-deficient mice 29. Weber C, Springer TA. Neutrophil accumulation on activated, surface- develop intimal hyperplasia after vascular injury: correlation with leu- adherent platelets in flow is mediated by interaction of Mac-1 with kocyte recruitment to adherent platelets 1 hour after injury. Circulation. fibrinogen bound to alphaIIbbeta3 and stimulated by platelet-activating 2001;103:2501–2507. factor. J Clin Invest. 1997;100:2085–2093. 43. Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription factor 30. McEver RP. Adhesive interactions of leukocytes, platelets, and the in chronic inflammatory diseases. N Engl J Med. 1997;336:1066–1071. vessel wall during hemostasis and inflammation. Thromb Haemost. 44. Suzuki J, Ogawa M, Muto S, Itai A, Isobe M, Hirata Y, Nagai R. Novel 2001;86:746–756. IkB kinase inhibitors for treatment of nuclear factor-kB-related diseases. 31. Diacovo TG, Roth SJ, Buccola JM, Bainton DF, Springer TA. Neutrophil Expert Opin Investig Drugs. 2011;20:395–405. rolling, arrest, and transmigration across activated, surface-adherent 45. Baldwin AS Jr. Series introduction: the transcription factor NF-kappaB platelets via sequential action of P-selectin and the beta 2-integrin and human disease. J Clin Invest. 2001;107:3–6. CD11b/CD18. Blood. 1996;88:146–157. 46. Uwe S. Anti-inflammatory interventions of NF-kappaB signaling: poten- 32. Cerletti C, Evangelista V, de Gaetano G. P-selectin-beta 2-integrin cross- tial applications and risks. Biochem Pharmacol. 2008;75:1567–1579. talk: a molecular mechanism for polymorphonuclear leukocyte recruit- 47. Traber KE, Okamoto H, Kurono C, Baba M, Saliou C, Soji T, Packer L, ment at the site of vascular damage. Thromb Haemost. 1999;82:787–793. Okamoto T. Anti-rheumatic compound aurothioglucose inhibits tumor 33. Evangelista V, Manarini S, Sideri R, Rotondo S, Martelli N, Piccoli A, necrosis factor-alpha-induced HIV-1 replication in latently infected Totani L, Piccardoni P, Vestweber D, de Gaetano G, Cerletti C. Platelet/ OM10.1 and Ach2 cells. Int Immunol. 1999;11:143–150. polymorphonuclear leukocyte interaction: P-selectin triggers protein- 48. Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA. Possible tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of new role for NF-kappaB in the resolution of inflammation. Nat Med. PSGL-1 as a signaling molecule. Blood. 1999;93:876–885. 2001;7:1291–1297. 34. Seals DF, Courtneidge SA. The ADAMs family of metalloproteases: 49. Kanters E, Pasparakis M, Gijbels MJ, Vergouwe MN, Partouns-Hendriks multidomain proteins with multiple functions. Genes Dev. 2003;17:7–30. I, Fijneman RJ, Clausen BE, Förster I, Kockx MM, Rajewsky K, Kraal 35. Schlöndorff J, Becherer JD, Blobel CP. Intracellular maturation and G, Hofker MH, de Winther MP. Inhibition of NF-kappaB activation in localization of the tumour necrosis factor alpha convertase (TACE). macrophages increases atherosclerosis in LDL receptor-deficient mice. J Biochem J. 2000;347(Pt 1):131–138. Clin Invest. 2003;112:1176–1185. 36. Rizoli SB, Rotstein OD, Kapus A. Cell volume-dependent regulation of 50. Zaph C, Troy AE, Taylor BC, Berman-Booty LD, Guild KJ, Du Y, L-selectin shedding in neutrophils. A role for p38 mitogen-activated pro- Yost EA, Gruber AD, May MJ, Greten FR, Eckmann L, Karin M, Artis tein kinase. J Biol Chem. 1999;274:22072–22080. D. Epithelial-cell-intrinsic IKK-beta expression regulates intestinal 37. Soond SM, Everson B, Riches DW, Murphy G. ERK-mediated phos- immune homeostasis. Nature. 2007;446:552–556. phorylation of Thr735 in TNFalpha-converting enzyme and its potential 51. Pahl HL. Activators and target genes of Rel/NF-kappaB transcription role in TACE protein trafficking. J Cell Sci. 2005;118(Pt 11):2371–2380. factors. Oncogene. 1999;18:6853–6866.

Downloaded from http://atvb.ahajournals.org/ at Georgia Regents University Greenblatt Library on December 12, 2013 Supplemental materials

Platelet IKKβ Deficiency Increases Mouse Arterial Neointima Formation via Delayed Glycoprotein Ibα Shedding

Shujian Wei, MD, PhD; Huan Wang, MD, PhD; Guoying Zhang, MD; Ying Lu, PhD; Xiaofei An, MD, PhD; Shumei Ren MD, PhD; Yunmei Wang; Yuguo Chen, MD, PhD; James G White; MD; Chunxiang Zhang, MD, PhD; Daniel I. Simon, MD; Chaodong Wu, MD, PhD; Zhenyu Li, MD, PhD; and Yuqing Huo, MD, PhD

Address correspondence to: Yuqing Huo, MD, PhD Professor, Chief of Vascular Inflammation Program Vascular Biology Center Department of Cellular Biology and Anatomy Medical College of Georgia Georgia Health Sciences University Sanders Building, CB-3919A 1459 Laney Walker Blvd Augusta, GA 30912-2500 Phone: 706-721-4414 (Office) Fax: 706-721-9799 (VBC) Email: [email protected]

Extended methods

Mice

PF4-cre 1 and floxed IKKβ mice 2 (C57BL/6 background) were received from Dr. Radek

C. Skoda and Dr. Michael Karin. The C57BL/6J and LDLR–/– mice were received from The

Jackson Laboratory (Bar Harbor, ME). PF4-cre and floxed IKKβ mice were crossed to generate platelet-specific IKKβ-deficient mice -IKKβfl/fl/PF4cre mice and their IKKβfl/fl littermates

(Supplemental Figure VII). A mouse 384 SNP panel (including markers spread across the genome at approximately 7 Mbp intervals) was used to characterize the genetic background of the breeding IKKβfl/fl/PF4cre mice (Charles River Laboratories International, Troy, NY).

IKKβfl/fl/PF4cre and LDLR–/– mice were crossed to generate IKKβfl/fl/PF4cre/LDLR–/– mice and their IKKβfl/fl/LDLR–/– littermates. Supplemental Figure I shows the breeding scheme. All of the animal experiments and care were approved by the Georgia Health Sciences University Animal

Care and Use Committee, in accordance with AAALAC guidelines.

Carotid artery wire injury model

Eight-week-old male mice were fed a Western diet (42.0% kcal fat, 42.7% kcal carbohydrate, 15.2% kcal protein) (Harlan Teklad, Madison, WI) for 2 weeks. A guide wire injury was then administered to the carotid artery.3-5 To quantify the neointimas, each carotid artery was serially sectioned from the bifurcation to the common carotid artery, resulting in 100 slides; each slide had 3 serial sections. Among these slides, 12 slides, including slides 1, 10, 19,

28, 37, 46, 55, 64, 73, 82, 91 and 100, were stained with Movat pentachrome (Sigma, St. Louis,

MO). For each carotid artery, 10 sections (one section on each above slide) were analyzed. The areas of the lumen, internal elastic lamina, and external elastic lamina were determined by planimetry using NIH Image Software, and the intimal area, medial area and intima to media

(I/M) ratio were calculated.3-5

Neutrophil depletion

After being fed a Western diet for 2 weeks, a carotid artery wire injury was performed on the IKKβfl/fl/PF4cre/ LDLR–/– mice and the IKKβfl/fl/ LDLR–/– control mice. A neutrophil-depleting anti-PMN antibody (Accurate Chemical & Scientific, Westbury, NY) was diluted in PBS (1:5), and 0.1 ml of the diluted antibody was injected into the mouse peritoneal cavity daily for 4 weeks after the wire injury.6 For the control mice, rabbit IgG (Accurate

Chemical & Scientific) was injected at the same dose. The efficacy of the anti-PMN antibody on

2 neutrophil depletion was confirmed by flow cytometry and HESKA CBC-Diff Veterinary

Hematology System (data not shown).

Bone marrow transplantation

The bone marrow transplantation was performed as previously described.5 Bone marrow-derived cells from the IKKβfl/fl/PF4cre/LDLR–/– mice or IKKβfl/fl/LDLR–/– controls were transplanted into lethally irradiated LDLR–/– mice. Three weeks after the bone marrow transplantation, the mice were fed a Western diet for 2 weeks and then subjected to carotid artery wire injury.

Histological analysis of the injured arteries

Cross sections of the injured arteries were stained with monoclonal antibodies to identify the platelets (MWReg30; Santa Cruz Biotechnology, Santa Cruz, CA), macrophages (F4/80, clone CI:A3-1; Accurate Chemical, Westbury, NY) and neutrophils (anti-mouse neutrophil, clone 7/4; Accurate Chemical). The images were analyzed with Image-Pro Plus.

In vivo examination of leukocyte interactions with the injured arterial wall

The left carotid arteries of the mice were injured with a guide wire to induce vascular damage. After 10 min, the leukocyte interactions with the injured vessel were recorded and analyzed with an intravital epifluorescence microscopy system.4

Leukocyte interactions with activated platelets in a micro-flow chamber

3 The mouse platelets were isolated from the IKKβfl/fl/PF4cre mice and their littermate

IKKβfl/fl mice by gel filtration.7 The micro-flow chamber was prepared as previously described.4,

5 Specifically, 2 x 109/ml of isolated platelets were loaded into a rectangular glass capillary tube.

After adhering to the surface of the capillary tube, the platelets were activated with thrombin (0.1

U/ml). The wild type mice were anesthetized and injected with 1 mg/ml rhodamine 6G (50 µl/30 g body weight) via the tail vein. Then, the blood from the carotid artery was passed through the micro-flow chamber. For some experiments, the mice were first treated with anti-M2 antibody and IgG. Leukocyte rolling and adhesion on the activated platelets was observed and recorded with an intravital epifluorescence microscope system.

Blocking of the GPIbα binding site on Mac-1

An affinity purified peptide-specific polyclonal antibody (termed anti-M2) to the Mac-1 binding site for GPIbα was kindly supplied by Dr. Daniel I. Simon.8 A total of 100 µg anti-M2 or

100 µg IgG (Accurate Chemical & Scientific) was injected via the tail vein of IKKβfl/fl/PF4cre/

LDLR–/– mice and the IKKβfl/fl/ LDLR–/– control mice that had been fed a Western diet for 2 weeks. Following antibody or IgG treatment, these mice were subjected to wire injury.

Platelet activation, aggregation and secretion

The mouse platelets were isolated by gel filtration.7 The purity of the platelets was determined by flow cytometry (Supplemental Figure VIII). Platelet activation was achieved by treating the platelets with thrombin (Sigma, St. Louis, MO) at the indicated concentrations, followed by neutralization with an equimolar dose of hirudin (Sigma) if necessary. As previously described,9 platelet aggregation was measured in a Lumi-Aggregometer model 700 (Chronolog)

4 at 37°C with stirring (1000 rpm). In parallel with platelet aggregation, platelet secretion was monitored as ATP release with the addition of the luciferin-luciferase reagent to the platelet suspension. Quantification was performed using ATP standards.

Flow cytometric analysis

The anti-Ly6G, anti-CD41 and anti-P-selectin antibodies were purchased from BD

Biosciences. The anti-CD115 antibody was from eBioscience (San Diego, CA). The anti-GPIbα, anti-GPV, anti-GPVI, anti-GPIX and anti-αIIbβ3 antibodies were obtained from Emfret

Analytics (Wurzburg, Germany). For whole blood flow cytometry, Ly6G and CD115 were used to identify mouse circulating neutrophils and monocytes, respectively. CD41 was used to detect platelets. Flow cytometry was performed using a FACSCalibur (BD Biosciences). The data were analyzed using CellQuest (Tampa, FL) software.

Platelet electron microscopy

Platelets was isolated from whole blood and fixed in glutaraldehyde in White’s saline. The platelets were then placed in 1% osmic acid in Zetterquiest’s buffer, dehydrated with alcohol, and embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate to enhance the contrast, and the sections were then examined with a Philips 301 electron microscope (F.E.I. Co., Hillboro, OR, USA).

Western blot analysis

The platelets were lysed in a modified RIPA lysis buffer.4 Primary antibodies against

GPIbα (Emfret Analytics, Wurzburg, Germany), ADAM17, active ADAM17 (Abcam,

5 Cambridge, MA), P38 MAPK, p-P38 (phosphorylated P38 MAPK), and GAPDH (Cell

Signaling, Danvers, MA) were added, followed by incubation with alkaline phosphatase–conjugated secondary antibodies. The membranes were developed with a chemiluminescence reagent. The band intensities were quantified using the NIH Image J program.

Blood lipid and leukocyte analysis

Plasma triglycerides, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and total cholesterol were measured via an automated enzymatic technique (Boehringer Mannheim

GmbH). Total and differential leukocyte counts in the blood were quantified using an automated blood cell counter (Hemavet 850FS, CDC Technologies, Oxford, CT).

Statistical analysis

The data are presented as the mean ± SEM. The data were analyzed by either one-way

ANOVA followed by a Bonferroni correction post-hoc test or Student’s t-test to evaluate two-tailed levels of significance. The null hypothesis was rejected at P < 0.05.

6 Supplemental Data

Supplemental Table I and II. Blood cell analysis (Table I) and lipid profile (Table II) of the

IKKβfl/fl/PF4cre/LDLR-/- mice and the control IKKβfl/fl/LDLR-/-mice

7

Supplemental Figure I

Supplemental Figure I. The breeding scheme of experimental mice.

8

Supplemental Figure II

Supplemental Figure II. The size of the neointima and media of the injured carotid arteries. The arterial neointima at 4 weeks after injury was stained with Movat pentachrome, and the ratio of intima (I) to media (M) was calculated (I/M, n = 12 for each group)

9 Supplemental Figure III

Supplemental Figure III. Platelet IKKβ deficiency increased neointima formation in the wire-injured carotid arteries from the LDLR-/- mice following bone marrow transplantation. The LDLR–/– mice were lethally irradiated and then received bone marrow from the IKKβfl/fl/PF4cre/LDLR–/– or IKKβfl/fl/LDLR–/– mice. Three weeks after bone marrow transplantation, the mice were fed a Western diet for 2 weeks and then subjected to carotid artery wire injury. A, Movat pentachrome staining of cross sections of the injured artery at 4 weeks after injury. The size of the neointima (I) and media (M) and the ratio of the intima to media (I/M) were quantified (n = 10 for each group). B, The infiltrated macrophages were immunostained with anti-F4/80 antibody in the arterial neointima. The percentage of the area that was positive was calculated by dividing the positive area by the measured lesion area (n = 10 for each group).

10 Supplemental Figure IV

Supplemental Figure IV. Platelet IKKβ deficiency increased platelet-leukocyte aggregates in vivo. IKKβfl/fl/PF4cre/LDLR–/– and IKKβfl/fl/LDLR–/– mice were fed a Western diet for 4 weeks. Whole blood was collected from the submandible and used for flow cytometry. Platelet-neutrophil aggregates were defined as cells positive for both CD41 (platelet marker) and Ly6G (neutrophil marker), whereas platelet-monocyte aggregates were defined as cells positive for CD41 and CD115 (monocyte marker). Representative flow cytometry results showing platelet-neutrophil aggregates (A), levels of GPIbα for platelet- leukocyte aggregates (B and D) and platelet-monocyte aggregates (C) (n = 5 for each group).

11

Supplemental Figure V

Supplemental Figure V. Effects of IKKβ deficiency on GPV shedding in platelets. The platelets were isolated from the IKKβfl/fl/PF4cre and IKKβfl/fl mice and stimulated with thrombin at 0.1

U/ml. Representative flow cytometric histograms show the level of GPV (A), GPVI (B), GPIX

(C) and αIIbβ3 (D) on the activated platelets. Three separate experiments with different donors were performed.

12

Supplemental Figure VI

Supplemental Figure VI. Neutrophil depletion decreased neointima formation in wire-injured carotid arteries of both IKKβfl/fl/PF4cre/LDLR–/– mice and IKKβfl/fl/LDLR–/– mice. The mice were fed a Western diet for 2 weeks and then subjected to carotid artery wire injury. Control rabbit

IgG and anti-PMN antibody were intraperitoneally injected into these mice daily. Four weeks later, the carotid arteries were collected, and the size of the neointima and media was analyzed with Movat pentachrome staining. Ten mice were included in each group.*P<0.05 vs.

IKKβfl/fl/LDLR–/– mice treated with control IgG; #P<0.05 vs. IKKβfl/fl/PF4cre/LDLR–/– mice treated with control IgG.

13

Supplemental Figure VII

Supplemental Figure VII. Western blot of isolated platelets showing that PF4-cre effectively knocked out platelet IKKβ expression in IKKβfl/flPF4cre mice.

Supplemental Figure VIII

Supplemental Figure VIII. The purity of isolated platelets was determined by flow cytometry.

Representative data showing that more than 99% platelets are CD41 positive.

14 References

1. Tiedt R, Schomber T, Hao-Shen H, Skoda RC. Pf4-cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood. 2007;109:1503-1506 2. Pasparakis M, Courtois G, Hafner M, Schmidt-Supprian M, Nenci A, Toksoy A, Krampert M, Goebeler M, Gillitzer R, Israel A, Krieg T, Rajewsky K, Haase I. Tnf-mediated inflammatory skin disease in mice with epidermis-specific deletion of ikk2. Nature. 2002;417:861-866 3. An G, Wang H, Tang R, Yago T, McDaniel JM, McGee S, Huo Y, Xia L. P-selectin glycoprotein ligand-1 is highly expressed on ly-6chi monocytes and a major determinant for ly-6chi monocyte recruitment to sites of atherosclerosis in mice. Circulation. 2008;117:3227-3237 4. Wang H, Zhang W, Tang R, Hebbel RP, Kowalska MA, Zhang C, Marth JD, Fukuda M, Zhu C, Huo Y. Core2 1-6-n-glucosaminyltransferase-i deficiency protects injured arteries from neointima formation in apoe-deficient mice. Arterioscler Thromb Vasc Biol. 2009;29:1053-1059 5. Wang H, Zhang W, Tang R, Zhu C, Bucher C, Blazar BR, Geng JG, Zhang C, Linden J, Wu C, Huo Y. Adenosine receptor a2a deficiency in leukocytes increases arterial neointima formation in apolipoprotein e-deficient mice. Arterioscler Thromb Vasc Biol. 2010;30:915-922 6. Zernecke A, Bot I, Djalali-Talab Y, Shagdarsuren E, Bidzhekov K, Meiler S, Krohn R, Schober A, Sperandio M, Soehnlein O, Bornemann J, Tacke F, Biessen EA, Weber C. Protective role of cxc receptor 4/cxc ligand 12 unveils the importance of neutrophils in atherosclerosis. Circ Res. 2008;102:209-217 7. Huo Y, Schober A, Forlow SB, Smith DF, Hyman MC, Jung S, Littman DR, Weber C, Ley K. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein e. Nat Med. 2003;9:61-67 8. Wang Y, Sakuma M, Chen Z, Ustinov V, Shi C, Croce K, Zago AC, Lopez J, Andre P, Plow E, Simon DI. Leukocyte engagement of platelet glycoprotein ibalpha via the integrin mac-1 is critical for the biological response to vascular injury. Circulation. 2005;112:2993-3000 9. Li Z, Xi X, Gu M, Feil R, Ye RD, Eigenthaler M, Hofmann F, Du X. A stimulatory role for cgmp-dependent protein kinase in platelet activation. Cell. 2003;112:77-86

15