Fig 2: NOR-1 and cIAP2 are over-expressed in human AAA tissues.(a) NOR-1 and cIAP2 mRNA levels determined by real-time PCR in human AAA samples (black bars; n = 96) and human healthy aortic wall (Donors; white bars; n = 16). Data are expressed as mean ± s.e.m. *P < 0.01 vs. Donors; #P < 0.0001 vs. Donors. ß-actin was used as endogenous control. (b) Representative western blot showing NOR-1 and cIAP2 protein levels in these tissues. Levels of ß-actin were used as a loading control. (c) Positive statistical correlation between mRNA levels of NOR-1 and cIAP2 in patients and donors. The Spearman Rank Order Correlation after logarithmic transformation of data was applied (n = 112; r = 0.454; P < 0.000001). (d) Representative high magnification images showing the immunohistochemical analysis of NOR-1, and cIAP2 in consecutive sections from human healthy aortic wall samples (Donors; upper panels) and human AAA (middle panels). Large lower panels show low magnification images from AAA immunostaining. The position of the corresponding high power views squared. Bar = 50 µm (in high magnification images); Bar = 200 µm (in low magnification images). A: adventitia, m: media, and l: lumen.

Fig 3: NFAT regulates PD-1 expression induced by CD69 engagement in T cells. A Cyclosporine A reduces the expression of PD-1 + in T cells. JKCD69 cells were incubated with crosslinked anti-CD69 with or without cyclosporine A (CsA), or phorbol 12-myristate 13-acetate and ionomycin (PMA/Io) stimulated with or without CsA for 48 h. Data are mean ± SD (n = 3), and differences were determined by unpaired t test. * P < 0.05. B Cyclosporine A inhibits CD69-mediated NR4A3 induction. Relative expression of NR4A3 in JKCD69 incubated with anti-CD69 monoclonal antibody, with or without CsA and with CsA in resting cells for 4 h. Data are mean ± SD (n = 3), and differences were analysed with one-way ANOVA (Tukey post hoc test). * P < 0.05, ** P < 0.01. C Induction of NR4A1 is independent of NFAT blockade. Relative expression of NR4A1 in JKCD69 with anti-CD69 antibody, with or without CsA and with CsA in resting cells incubated for 4 h. Data are mean ± SD (n = 3) and were analysed with one-way ANOVA (Tukey post hoc test). *P < 0.05, **P < 0.01. D Relative expression levels of NR4A3 mRNA after siRNA knockdown in resting and anti-CD69 stimulation conditions. Data showed are mean ± SD (n = 3), and differences were determined by unpaired t test. *P < 0.05. ** P<0.01. E siRNA interfering of NR4A3 expression enhances PD-1 + cell population. JKCD69 were stimulated with anti-CD69 or PMA/Io for 48 h. Data are mean ± SD (n = 3), and differences were determined by paired t test. *P < 0.05

Fig 4: PD-1 and CD69 are highly expressed in inflamed human arteries. A PD-1 and CD69 mRNA levels were measured in inflamed abdominal aortic samples (IAA) from patients undergoing open repair surgery for abdominal aortic aneurysm (n = 49) and healthy abdominal aortic samples (HAA) from donors (n = 11). Data are expressed as mean ± SD *P < 0.05 (Mann–Whitney test). (right panel) Statistically significant positive correlation between CD69 mRNA levels and those corresponding to PD-1 in inflamed aortic samples from patients. The Pearson Product Moment Correlation was applied. B CD69, PD-1 and NR4A3 protein levels were evaluated by western blot in aortic lysates from IAA and HAA samples. Representative immunoblots are shown. C NR4A3 mRNA levels assessed in the samples described in A). Data are expressed as mean ± SD. *P < 0.05 (Mann–Whitney test). D Immunohistochemical analysis of CD69, PD-1 and NR4A3 in haematoxylin counterstained sections from IAA and HAA samples. The indicated areas are shown at high magnification (middle panels). Bar: 100 µm (upper and lower panels) and 50 µm (middle panels). Negative controls in which the primary antibody was omitted are shown on the right

Fig 5: NOR-1 and cIAP2 are modulated by CoCl2 in VSMC in a dose- and time-dependent manner.(a,b) NOR-1 (white bars) and cIAP2 (black bars) mRNA levels analyzed by real-time PCR in VSMC exposed to increasing concentrations of CoCl2 (a) or CoCl2 (0.5 mM) for increasing times (b) (n = at least 5). *P < 0.0001 vs. cells maintained in normoxia (Norm). (c) Representative western blot showing protein levels of NOR-1 and cIAP2 in VSMC exposed to CoCl2 (0.5 mM) for increasing times. Levels of ß-actin are shown as a loading control in western blot analysis. (d) Immunofluorescence confocal microscopy analysis showing cIAP2 (green) and nuclei (Hoechst stain, blue) in VSMC exposed to CoCl2 for 6 hours. Bar = 25 µm.