Fig 2: Identification of endothelial protein C receptor (EPCR) and scavenger receptor class B type 1 (SR-BI) as endothelial autoantigens in Takayasu arteritis.a HUVEC cDNA fragments inserted into the genomic DNA of C1 and C3 clones established with U10-4 IgG were amplified, and PCR products were electrophoresed on a 0.8% agarose gel. b DNA sequencing was performed for the PCR products obtained around 2000 bp for C1, followed by BLAST analysis. c C1 (left) and C3 (right) were stained with PE-conjugated isotype control or PE-conjugated anti-human EPCR antibody and analyzed with flow cytometry. d The expression vector EPCR-IRES-GFP was transfected into YB 2/0 cells, and the cells were stained with 0.5 mg/mL control IgG or U10-4 IgG, followed by incubation with secondary antibody and flow cytometry analysis. e Inhibition tests for binding activities to YB2/0 cells overexpressing EPCR were performed using 0.5 mg/mL U10-4 IgG with soluble recombinant EPCR at the indicated concentrations. f Western blotting of recombinant EPCR proteins was performed, and they were stained with control serum, U10-4 serum, or anti-human EPCR antibody, followed by secondary antibodies. g HUVEC cDNA fragments inserted into the genomic DNA of C6 clones established with W10-59 IgG and C7 by using G10-43 IgG were amplified, and PCR products were electrophoresed on a 0.8% agarose gel. h DNA sequencing was performed for the PCR products obtained around 3000 bp for C7, followed by BLAST analysis. i C6 (left) and C7 (right) were stained with anti-human SR-BI antibody or isotype control, followed by incubation with secondary antibody and flow cytometry analysis. j The expression vector SR-BI-IRES-GFP was transfected into YB 2/0 cells, and the cells were stained with 0.5 mg/mL control IgG, W10-59 IgG, or G10-43 IgG, followed by incubation with secondary antibody and flow cytometry analysis. k Inhibition tests for binding activities to YB2/0 cells overexpressing SR-BI were conducted using 0.5 mg/mL W10-59 IgG with soluble recombinant SR-BI at the indicated concentrations. l YB2/0 cells expressing with or without SR-BI were reacted with control, W10-59, or G10-43 serum. Cells were then lysed and immunoprecipitation was performed. Western blotting was then performed, and the membrane was analyzed for the expression of SR-BI and human IgG. m Nonpermeabilized HUVECs were stained with PE-conjugated anti-human SR-BI antibody or isotype control and analyzed with flow cytometry. G10-43, W10-59, and U10-4 indicate the serum sample number.

Fig 3: Blocking of anti-inflammatory activities of EPCR and SR-BI by autoantibodies in Takayasu arteritis.a, b HUVECs were treated with or without 10 µg/mL APC for 13 h and stimulated with 100 pg/mL TNF-a for 5 h. The expression of adhesion molecules, including E-selectin, VCAM-1, and ICAM-1, was analyzed with flow cytometry. Representative histograms (left) and the summary graph (right, n = 3) are shown in a. The mRNA expression level was measured by quantitative PCR in b. GAPDH was used as the internal control. c HUVECs were incubated with the isotype or 10 µg/mL anti-EPCR antibody for 1 h and treated with or without 10 µg/mL APC for 13 h. Then, cells were stimulated with 100 pg/mL TNF-a for 5 h. The expression of adhesion molecules was analyzed with flow cytometry. d HUVECs were incubated with or without IgG for 1 h. IgG included 10 µg/mL anti-EPCR antibody, 2.56 mg/mL control IgG, 2.56 mg/mL IgG from an AECA-negative TAK sample (L11-05), or 2.56 mg/mL IgG from anti-EPCR-positive TAK AECA sample (J11-14). The cells were subsequently treated as described above, and the expression of adhesion molecules was analyzed. e HUVECs were treated with or without 1 mg/mL high-density lipoprotein (HDL) for 16 h and stimulated with 100 pg/mL TNF-a for 5 h. Adhesion molecules were analyzed by flow cytometry; the summary graph is shown (n = 5). f HUVECs were incubated with or without IgG for 1 h. IgG included 10 µg/mL anti-SR-BI antibody, 2.56 mg/mL control IgG, or 2.56 mg/mL IgG from anti-SR-BI positive TAK AECAs (M11-36). Cells were subsequently treated with or without 1 mg/mL of HDL as described above, and adhesion molecules were analyzed (n = 3). Data are indicated as mean ± SD. At least three independent experiments were performed in all cases. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. MFI represents mean fluorescent intensity. L11-05, J11-14, and M11-36 indicate the serum sample number.

Fig 4: Expressions of EPCR and SR-BI in the vasa vasorum of the aorta.Immunohistochemical analysis was performed against EPCR (a) and SR-BI (b) in resected aortic tissue from three different patients with TAK (upper two columns) and non-inflammatory aortic tissue as disease controls (lower column). Disease controls included surgical specimen from patients with aortic aneurysm and aortic stenosis, and an autopsy sample from a patient with hypothyroidism. Expressions in the vasa vasorum of the aorta are shown. Black bar indicates 40 µm.