Fig 2: Proposed model for the pathogenicity of AECAs in Takayasu arteritis.a APC and HDL, the ligand of EPCR and SR-BI, respectively, suppress expression of adhesion molecules in activated endothelial cells. APC also negatively regulates the differentiation of CD4+ T cells expressing EPCR into Th17 cells, and thus inhibits the production of Th17-related cytokines, including IL-17A, IL-17F, IL-21, and IL-22. These mechanisms contribute to the resolution of vascular inflammation in normal condition. b Autoantibodies against EPCR or SR-BI block bindings of corresponding ligands to their receptors, which promote expression of adhesion molecules in endothelial cells. In addition, anti-EPCR autoantibodies disrupt the negative regulation of Th17 differentiation by APC and thus amplify Th17-mediated immune response. As a result, autoantibodies in TAK inhibit the spontaneous resolution of activated immune responses in the vessel wall and thus lead to the chronic vascular inflammation.

Fig 3: 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 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.

Fig 5: Lipid droplet associated proteins and lipid uptake receptor expression differ between mitotane-sensitive and -resistant cell lines. Western blotting data represents protein expression of (A) PLIN1, PLIN2, PLIN3, PLIN4, and B actin in H295R, MUC1, and HEPG2. (B) Graphical representation of relative mRNA expression of Plin2 and Plin3 in H295R and MUC-1 cell lines. (C) Graphical representation of lipid uptake receptor expression CD36, LDLR, SCARB1, and LRP1 in H295R and MUC-1. (D) Western blot data represents protein expression of HSL in H295R, MUC-1, and HEPG2. (E) Graphical representation of relative mRNA expression of Lipe H295R and MUC-1 cell lines Western blot data represents protein expression of (G) SOAT1 and (H) DGAT1 HSL in H295R, MUC-1, and HEPG2. For western blot analysis, ß actin was used as a normalizing protein. Images are representative of 3 independent blots, numerical annotation is representative of semiquantitative analysis using ImageJ relative to H295R. mRNA expression is represented as fold change relative to H295R, B2M was used as a normalizing gene. Data are represented as mean ± SEM (n = 3), statistical comparisons were performed using a 2-tailed t-test or a 2-way ANOVA followed by Tukey post hoc analysis. Statistical significance is denoted as actual P values relative to vehicle control or as otherwise indicated.