Disturbed shear stress reduces the expression of AFF3ir-ORF2 in vivo and in vitro.

A–F, Aortas were isolated from the C57BL/6 mice. A, RT-PCR analysis of the mRNA levels of AFF3ir-ORF2 and AF4/FMR2 family member 3 (AFF3) in the thoracic aorta (TA) and aortic arch (AA) of C57BL/6 mice. Data are presented as mean ± SEM (n=6 mice per group). *P<0.05, unpaired two-tailed t-test. B, C, Western blot analysis of the expression of the indicated proteins in the TA and AA of mice. Protein levels were normalized to those of GAPDH, and the relative expression values were relative to those of the TA group. Data are presented as mean ± SEM (n=6 mice per group). *P<0.05, unpaired two-tailed t-test. D, E, En-face immunofluorescence staining of AFF3ir-ORF2, VE-cadherin, and DAPI, and quantification of AFF3ir-ORF2 expression in AA and TA. Scale bar, 20 μm. The immunofluorescence intensity of AFF3ir-ORF2 was normalized to that of DAPI, and the relative expression of the values was relative to that of the TA group. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test. F, Representative immunofluorescent staining for von Willebrand factor (vWF), AFF3ir-ORF2, and AFF3 in longitudinal sections of mouse aortas. n=6 mice per group. Scale bar, 25 μm. Inner, inner curvature of the AA; outer, outer curvature of the AA; BIF, Bifurcation. G, H, Mouse embryonic fibroblasts (MEFs) isolated from the embryo of C57BL/6 mice were subjected to static (ST) or oscillatory shear stress (OSS, 0.5 ± 4 dyn/cm2, 1 Hz) for indicated time. Western blot analysis of the indicated proteins. Protein levels were normalized to GAPDH and the relative expression values were relative to that of the ST group. Data are mean ± SEM (n = 6 independent experiments). *P< 0.05, one-way ANOVA with Tukey post-test. I, MEFs were subjected to ST or OSS treatment for 6 h. RT-PCR analysis of the mRNA levels of AFF3ir-ORF2, AFF3, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in MEFs. Data are mean ± SEM (n = 6 independent experiments). *P< 0.05, unpaired two-tailed t-test.

AFF3ir-ORF2 overexpression alleviates disturbed flow-induced inflammation and atherosclerosis.

A–C, Mouse embryonic fibroblasts (MEFs) isolated from C57BL/6 mice were infected with indicated adenoviruses (Ad-Scramble or Ad-Aff3ir-ORF2) for 48 h and then exposed to static (ST) or oscillatory shear stress (OSS, 0.5 ± 4 dyn/cm2, 1 Hz) for another 6 h. A, B, Western blot analysis of the indicated proteins in MEFs and quantification of their relative expression of proteins are shown. The protein levels were normalized to GAPDH and the relative expression of the values is relative to MEFs infected with Ad-Scramble and treated with ST. Data are presented as mean ± SEM (n = 6 independent experiments). *P< 0.05, two-way ANOVA with Tukey post-test. C, RT-PCR analysis of mRNA levels of AFF3, AFF3ir, VCAM-1, ICAM-1, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). The relative expression of the values is relative to MEFs infected with Ad-Scramble and treated with ST. Data are presented as mean ± SEM (n = 6 independent experiments). *P< 0.05, two-way ANOVA with Tukey post-test. D–H, Eight-week-old male ApoE−/− mice were subjected to partial ligation of the carotid artery along with 10 μL of adenovirus suspension at 1 × 108 transducing units (TU)/mL was instilled into the left carotid artery (LCA). The mice were then fed high-fat diet for 4 weeks. D, Arterial tissues were isolated to examine the atherosclerotic lesions. Scale bar, 2 mm. E, F, LCAs were sectioned for hematoxylin and eosin staining and quantification of the lesion area. Scale bar, 25 μm. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test. G, H, Immunofluorescence staining for vWF, VCAM-1, and DAPI in the LCA, and quantification of the relative fluorescent intensity of VCAM-1. The immunofluorescence intensity of VCAM-1 was normalized to DAPI, and the relative expression of the values were relative to that of the Ad-Scramble group. Scale bar, 50 μm. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test.

AFF3ir-ORF2 deletion aggravates inflammation and atherosclerotic lesions in ApoE-/- mice.

Eight-week-old male ApoE-/- and ApoE-/-AFF3ir-ORF2-/- mice were fed a high-fat diet for 12 weeks. Arterial tissues and aortic roots were isolated to examine atherosclerotic lesions. A, Schematic of experimental strategy. B, Representative images of en face Oil-Red O staining of the aortas. Scale bar, 4 mm. C, Quantification of the plaque area in the aortas. Data are presented as mean ± SEM (n=12 mice per group). *P< 0.05, unpaired two-tailed t-test. D, Oil-Red O, hematoxylin and eosin (HE), and Masson staining of the aortic roots. Scale bars, 500 μm. E, Quantification of plaque size, Oil-Red O-positive area, and collagen fiber content in aortic root sections. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test. F, LCAs were sectioned and stained with Oil-Red O, HE, and Masson’s trichrome. Scale bars, 500 μm. G, Quantification of plaque size, Oil-Red O-positive area, and collagen fiber content in the LCA sections. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test. H: Representative immunofluorescence images of vWF, VCAM-1, and DAPI in the aortic roots. Scale bar, 500 μm. I, Quantification of the relative fluorescence intensity of VCAM-1. The immunofluorescence intensity of VCAM-1 was normalized to that of DAPI, and the relative expression of the values were relative to that of the ApoE-/- group. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test.

AFF3ir-ORF2 mitigates disturbed shear stress-induced inflammation by interacting with IRF5 and retaining it within the cytosol.

A–D, Mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) and AFF3ir-ORF2-/- mice. A, Heat map showing mRNA profiles of WT and AFF3ir-ORF2-/- MEFs (n=3). B, Volcano map displays the differentially expressed genes between the groups. C, Gene Ontology enrichment pathway analysis of the differentially expressed genes. D, Venn diagrams of the top 20 transcription factors from the ChEA3 and DisGENET analysis related to atherosclerosis. E, Immunoprecipitation performed using antibodies against AFF3ir-ORF2, IRF5, and IRF8. n = three independent experiments. F–H, WT and AFF3ir-ORF2-/- MEFs were subjected to silence of Control (siNC), IRF5 (siIRF5), or IRF8 (siIRF8) with siRNAs for 24 h, followed by exposure to Static (ST) or oscillatory shear stress (OSS, 0.5 ± 4 dyn/cm2, 1 Hz) for another 6 h. F, RT-PCR analysis of the mRNA levels of VCAM-1, ICAM-1, IL-6, and IL-1b. The relative expression of the values is relative to WT MEFs transfected with siNC and treated with ST. Data are mean ± SEM (n = 6 independent experiments). *P< 0.05, two-way ANOVA with Tukey post-test. G, H, Representative western blots of IRF5 and ICAM-1 expression. Data are mean ± SEM (n = 5 independent experiments). *P< 0.05, two-way ANOVA with Tukey post-test. I, J, WT, and AFF3ir-ORF2-/- MEFs were exposed to ST or OSS for 6 h. Nuclear and cytoplasmic proteins were extracted from the cells. Representative western blots of the indicated proteins and quantification of IRF5 expression in nucleus are shown. The expression of these proteins was relative to the level of nuclear IRF5 in ST-treated WT MEFs. Data are mean ± SEM (n = 6 independent experiments). *P< 0.05, two-way ANOVA with Tukey post-test. K, HEK293 cells were transfected with the firefly luciferase reporter plasmid containing the IRF5-responsive ZNF217 promoter along with a β-galactosidase reporter plasmid for 24 hours. Cells were infected with the indicated adenoviruses (Ad-Scramble or Ad-Aff3ir-ORF2) for 24 h. Promoter activity was measured using luciferase, which was normalized to β-gal. Data are mean ± SEM (n = 6 independent experiments). *P< 0.05, two-way ANOVA with Tukey post-test.

IRF5 knockdown prevents the aggravation of atherosclerosis in AFF3ir-ORF2 deficienct mice.

Eight-week-old male ApoE-/- mice were subjected to partial ligation of the left carotid artery (LCA) along with 10 μL of lentivirus suspension at 1 × 108 transducing units (TU)/mL was instilled into the LCA. The mice were then fed a high-fat diet for 4 weeks. A, Arterial tissues were isolated to examine the atherosclerotic lesions. Scale bar, 2 mm. B, LCAs were sectioned for hematoxylin and eosin staining and quantification of the lesion area. Scale bar, 25 μm. Data are mean ± SEM (n=6 mice per group). *P< 0.05, two-way ANOVA with Tukey post-test. C, D, Immunofluorescence staining for vWF, VCAM-1, and DAPI in the LCAs and quantification of the relative fluorescent intensity of VCAM-1. Scale bar, 50 μm. The immunofluorescence intensity of VCAM-1 was normalized to DAPI, and the relative expression of the values were relative to that of the group of ApoE-/- mice infected with Ad-scramble. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, two-way ANOVA with Tukey post-test.

Endothelial-specific AFF3ir-ORF2 supplementation alleviates EC activation and atherosclerosis in ApoE-/- mice.

Eight-week old ApoE-/- male mice were infused with the indicated adeno-associated virus (AAV) and then fed a high-fat diet for 12 weeks. A, Schematic of the experimental strategy. B, Representative images of enface Oil-Red O staining of the aortas. Scale bar, 4 mm. C, Quantification of the plaque area in the entire aortas. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test. D, Hematoxylin and eosin (HE), Oil-Red O, and Masson staining of the aortic roots. Scale bars, 500 μm. E, Quantification of plaque size, Oil-Red O-positive area, and collagen fiber content in aortic root sections. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test. F, Representative immunofluorescence image of vWF, VCAM-1, and DAPI in the aortic roots. Scale bar, 500 μm. G, Quantification of the relative fluorescent intensity of VCAM-1. The immunofluorescence intensity of VCAM-1 was normalized to that of DAPI, and the relative expression of the values were relative to that of the AAV-Scramble group. Data are presented as mean ± SEM (n=6 mice per group). *P< 0.05, unpaired two-tailed t-test.

Schematic illustration of the AFF3ir-ORF2/IRF5 cascade in disturbed flow-induced endothelial activation and atherosclerosis.

Disturbed flow induced a down-regulation of AFF3ir-ORF2, which could interact with IRF5 and promote the latter’s retention in the cytoplasm, thereby boosting IRF5-dependent inflammatory pathways in endothelial cells and leading to atherogenesis.