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Supporting Information: Teaching Materials; Figs 1–5 as PowerPoint slide.

Figure S1 Characterization of primary culture of endothelial cells from human carotid artery and morphological change and COX-2 induction upon shear stress in these cells. (A) A phase-contrast image of cultured endothelial cells. Cells showed the cobblestone-like appearance, which was a character of endothelial cells. Bar = 300 µm. (B) Western blot analysis of cultured endothelial cells (ECs) from human carotid artery and cell lines derived from endothelial cells (aortic EC) and smooth muscle cells (aortic SMC). α-smooth muscle actin (SMA), a marker for smooth muscle cells, and von Willebrand Factor (vWF), a marker for endothelial cells were analysed. α-Tubulin was served as an internal control. (C) Immunostaining for VE-Cadherin (CD144), a marker for endothelial cells. Bar = 50 µm. (D) Uptake of FITC-labeled acetyl-LDL. Cells were incubated with FITC-labeled acetyl-LDL for 4 h. Bar = 50 µm. The above data verified the endothelial profile of these primary cells. (E) Phase-contrast images of endothelial cells without (0 Pa) or with (1.5 Pa) shear stress. An arrow shows the direction of shear stress. Cells were stretched upon shear stress, compared with a cobblestone-like appearance without shear stress. Bar = 100 µm. (F) Immunostaining for COX-2 (green) without or with shear stress. DAPI was used for nuclear staining (blue). Shear stress induced COX-2 expression in endothelial cells.

Figure S2 Characterization of COX-2 and EP2 expressing cells in human CA walls. (A) Double immunostaining for COX-2 (black) with either von Willebrand factor (vWF, red), a marker for endothelial cells, or α- smooth muscle actin (SMA, red), a marker for smooth muscle cells. (B) Double immunostaining for EP2 (black) with the same markers as in (A). Bar = 20 µm. Note that COX-2 and EP2 were expressed in both endothelial cells and smooth muscle cells.

Figure S3 Induction of COX-2 and mPGES1 in rat CA walls and characterization of COX-2 expressing cells. (A) Immunostaining for either COX-2 or COX-1 (green) with a-smooth muscle actin (red), a marker for smooth muscle cells, before (0 M) and at 3 months after (3 M) CA induction. Adjacent sections stained by Elastica van Gieson staining (EvG) are also shown. Representative images (right) were taken from an area indicated by the box in the schema of the arterial bifurcation (left). COX-2 expression was induced during CA formation, while COX-1 expression was not altered. Bar = 20 µm. ACA: anterior cerebral artery, ICA: internal carotid artery, OA: olfactory artery. (B) Double immunostaining for COX-2 (green) and either CD68 (for macrophage, red), endothelial nitric oxide synthase (eNOS for endothelial cells, red) or α-smooth muscle actin (SMA for smooth muscle cells, red). COX-2 was expressed in all the examined cell types, especially in macrophages and endothelial cells. Bar = 20 µm. (C) Proportions of CD68-positive, eNOS-positive or SMA-positive cells in COX-2 expressing cells (n = 5). Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test. *P < 0.05. (D) Immunostaining for mPGES1, mPGES2 or cPGES at the arterial bifurcation before (0 M) and at 3 months after (3 M) CA induction. Whereas these isoforms were constitutively expressed in this region, mPGES1, but not cPGES nor mPGES2, was up-regulated during CA formation. Note that mPGES1 induction was most prominent at the neck portion of CA (arrows). Bar = 50 µm.

Figure S4 Western blot analysis and immunostaining for EP2 expression during CA formation. (A) Western blot analysis for EP2. Proteins were extracted from the circle of Willis of rats before (0 M) and at 1 month (1 M) and 3 months after (3 M) CA induction and subjected to Western blot analysis (left). A representative blot from 5 independent experiments is shown. EP2 was induced in cerebral arteries during CA formation. The specificity of EP2 antibody was confirmed using samples from wild-type (Ptger2+/+) and Ptger2-deficient mice (Ptger2−/−) (right). α-Tubulin was served as an internal control. (B) Immunostaining for EP2. EP2 (green) and α-smooth muscle actin (red), a marker for smooth muscle cells, were stained at the arterial bifurcation before (0 M) and at 1 month (1 M) and 3 months after (3 M) CA induction (left). Bar = 20 µm. EP2 signals were increased during CA formation. EP2 signals were abolished in the tissue from Ptger2-deficient mice (Ptger2−/−), confirming the specificity of EP2 antibody (right). Bar = 10 µm.

Figure S5 The architecture of the circle of Willis and systemic blood pressure after CA induction in Ptger2-deficient mice. (A) The architecture of the circle of Willis from wild-type (Ptger2+/+) and Ptger2-deficient mice (Ptger2−/−). No apparent difference was observed between the two genotypes. MCA: middle cerebral artery. Other abbreviations are the same as in Supporting Information Figure S3. (B) The systemic blood pressure (systolic blood pressure) of mice deficient of each EP subtypes (Ptger1−/−, Ptger2−/−, Ptger3−/−, Ptger4−/−), Ptger2-heterozygous mice (Ptger2+/−), and wild-type mice (WT) at 5 months after CA induction. Systemic blood pressure was similar across the genotypes. Numbers of animals are shown in parentheses. Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test. *P < 0.05 compared with the value of wild-type mice.

Figure S6 The contents of PGI2 and thromboxane A2 metabolites in CA walls and the incidence of CA formation in mice deficient in either Tbxa2r or Ptgir. (A) The contents of 6-keto-PGF and TxB2, stable metabolites of PGI2 and TXA2, respectively, in the arterial bifurcation before (0 M) and at 1 month (1 M) and 3 months after (3 M) CA induction. The content was measure by ELISA and normalized to the wet weight of tissues. The contents of these metabolites were also measured in rats at 3 months after sham operation (sham). Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test (n = 5). *P < 0.05, **P < 0.01. (B) The incidence of CA formation in mice deficient either Tbxa2r (Tbxa2r−/−) or Ptgir (Ptgir−/−) and wild-type mice (WT). No significant difference was observed in Tbxa2r−/−or Ptgir−/− mice compared to wildtype mice. Numbers of animals are shown in parentheses. Incidence was analysed by Fisher’s exact test.

Figure S7 Effects of celecoxib treatment on chronic inflammation in CA walls. (A) Systemic blood pressure (systolic blood pressure) of rats treated with celecoxib. Rats were treated with celecoxib or vehicle at 150 mg·kg−1·day−1 for 3 months after CA induction. The systemic blood pressure was measured by the tail-cuff method. Numbers of animals are shown in the parentheses. Data were analysed using Mann–Whitney U-test. (B) The number of CD68-positive macrophages in CA walls. CD68-positive macrophages were identified by immunostaining. Numbers of animals are shown in parentheses below each bar. Celecoxib treatment significantly inhibited macrophage infiltration in CA walls. Data were analysed using Mann–Whitney U-test. *P < 0.05. (C) Effects of celecoxib on mRNA levels of Mmp2, Ccl2 and Il-1β at the arterial bifurcation before (0 M) and after (3 M) CA induction. mRNA levels were determined by quantitative RT-PCR analysis (n = 6). Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test. *P < 0.05, **P < 0.01. (D) Immunostaining for MMP2, CCL2 and IL-1β in CA walls of rats treated without or with celecoxib. Green signals show MMP2, CCL2 or IL-1β in the corresponding images. Red signals show the signal for α-smooth muscle actin. Bar = 20 µm. (E) The effect of celecoxib treatment on gelatinase activity of MMP2 in cerebral arteries of rats during CA formation. Cerebral arteries from sham-operated rats (sham) or CA-induced rats without or with celecoxib treatment were subjected to gelatin zymography. A representative image (upper) and average signal intensities of MMP2 band from five independent experiments (lower) are shown. Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test. *P < 0.05, **P < 0.01. (F) The effect of celecoxib on NF-κB phosphorylation. The phosphorylated form of NF-κB p65 was immunostained in CA walls without or with celecoxib (left), and the proportion of positive cells was quantified (right) (n = 5). DAPI was used for nuclear staining (blue). Data were analysed using Mann–Whitney U-test. **P < 0.01. Celecoxib treatment significantly suppressed NF-κB activation. (G) Immunostaining for COX-2 and the phosphorylated form of NF-κB p65 subunit (p-p65) in rat CA walls at 3 months after CA induction. These two signals were mostly co-localized. Bar = 20 µm. (H) The effect of NF-κB decoy ODN on COX-2 expression in CA wall. Rats were treated with NF-κB decoy ODN and scrambled decoy ODN for 3 months after CA induction. COX-2 and the α-smooth muscle actin are shown in green and red, respectively. Bar = 30 µm. NF-κB inactivation by its decoy ODN suppressed COX-2 expression.

Figure S8 The effect of an EP receptor agonist on NF-κB phosphorylation and CCL2 expression and EP2 knockdown by RNAi in endothelial cells from human carotid artery. (A) Western blot analysis for the phosphorylated form of NF-κB p65 subunit (p-p65) and CCL2 expression in endothelial cells treated with 16-16-dimethyl PGE2, an EP receptor agonist, or vehicle for 24 h. Representative blots from 5 independent experiments are shown. α-Tubulin was served as an internal control. (B) The quantification by the densitometric analysis for NF-κB phosphorylation (p-p65, left) and CCL2 (right) (n = 5). This EP agonist induced NF-κB phosphorylation and CCL2 expression in a dose-dependent manner. Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test. *P < 0.05, **P < 0.01. (C) Western blot analysis (left) and immunostaining (right) for EP2 in primary culture of endothelial cells. Bar = 50 µm. (D, E) EP2 depletion by RNAi. Cells were transfected with scrambled (control) or human Ptger2 siRNA and incubated for 72 h. Quantitative RT-PCR analysis (D, n = 6) and Western blot analysis (E, n = 5) were performed. α-Tubulin was served as an internal control. Data were analysed using Mann–Whitney U-test. *P < 0.05, **P < 0.01.

Figure S9 Induction of iNos and Il-1β mRNA by an EP2 agonist in the primary culture of smooth muscle cells. (A) Induction of iNos and Il-1β mRNA by EP agonists. Primary culture of smooth muscle cells was incubated with agonists selective to each EP subtypes (EP1 to EP4) at 0.5 µM and vehicle for 24 h. mRNA levels were determined by quantitative RT-PCR analysis (n = 6). Only an EP2 agonist induced iNos and Il-1β mRNA expression. Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test. *P < 0.05, **P < 0.01. (B) A dose-dependent effect of an EP2 agonist on iNos and Il-1β mRNA expression (n = 6). Data were analysed using Kruskal–Wallis test followed by post hoc Dunn’s test. *P < 0.05, **P < 0.01.

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BPH_1358_sm_Figs1-5.pptx682KSupporting info item
BPH_1358_sm_FigureS1.tif3765KSupporting info item
BPH_1358_sm_FigureS2.tif1704KSupporting info item
BPH_1358_sm_FigureS3.tif3570KSupporting info item
BPH_1358_sm_FigureS4.tif2201KSupporting info item
BPH_1358_sm_FigureS5.tif213KSupporting info item
BPH_1358_sm_FigureS6.tif123KSupporting info item
BPH_1358_sm_FigureS7.tif4298KSupporting info item
BPH_1358_sm_FigureS8.tif1909KSupporting info item
BPH_1358_sm_FigureS9.tif128KSupporting info item

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