Fig 2: Soluble TWEAK is superior to TNF in TRAF1 mRNA induction. (A,B) TRAF1 expression was induced in the indicated cell lines by overnight stimulation with Flag-TWEAK (200 ng/ml) or Flag-TNF (100 ng/ml). Cells were then treated for additional 4 h with the proteasome inhibitor MG132 (20 µM) and/or the protein synthesis inhibitor cycloheximide (25 µg/ml). (B) HT1080 cells were stimulated overnight with TNF and TWEAK in the presence and absence of z-VAD-fmk (20 µM) and total cell lysates were analyzed by western blotting for TRAF1 induction. (C,D) Total RNA was isolated from cells stimulated with Flag-TNF (100 ng/ml) or Flag-TWEAK (200 ng/ml) overnight and subjected to analysis by reverse transcriptase PCR (C) and quantitative real time PCR. The average fold induction calculated from normalized data derived of three fully independent experiments is shown (D).

Fig 3: TWEAK or Fn14 deletion diminished pro-inflammatory chemokine expression in abdominal aortic aneurysm. A, Representative staining and (B) quantification of positive staining of Ki-67 in AAA lesions from TWEAK KO (N=7), Fn14 KO (N=7) and WT (N=7) mice at 14 days postperfusion. Values shown are mean±SE. Negative controls were incubated with non-specific IgG. Scale bar, 50 µm. C, Representative staining and (D) quantification of positive staining of CCL2 and CCL5 in AAA lesions from TWEAK KO (N=9), Fn14 KO (N=9) and WT (N=10) mice at 14 days postperfusion. Values shown are mean±SE. *P<0.005 vs WT. Negative controls were incubated with non-specific IgG. Scale bar, 50 µm. E, Effect of TWEAK on CCL2 and CCL5 secretion in cultured VSMC. WT or Fn14 KO VSMC were incubated with 0.01 to 0.1 µg/mL TWEAK during 24 hours and supernatants were tested by ELISA for CCL2 or CCL5. Values shown mean±SE. *P<0.005 vs basal. F, Effect of TWEAK on CCL2 and CCL5 secretion in cultured BMDM. WT or Fn14 KO VSMC were incubated with 0.01 to 0.1 µg/mL TWEAK during 24 hours and supernatants were tested by ELISA for CCL2 or CCL5. Values shown mean±SE. *P<0.01 vs basal. AAA indicates abdominal aortic aneurysm; BMDM, bone marrow-derived macrophages; CCL2, Chemokine Ligand 2; CCL5, Chemokine Ligand 5; ELISA, enzyme-limked immunosorbent assay; KO, knockout; MCP-1, Monocyte Chemoattractant Protein 1; RANTES, Regulated on Activation, Normal T-cell Expressed and Secreted; TW, TWEAK; TWEAK, tumor necrosis factor-like weak inducer of apoptosis; VSMC, vascular smooth muscle cells; WT, wild type.

Fig 4: TWEAK or Fn14 deletion diminished elastin degradation and MMP-activity in abdominal aortic aneurysm. A, Representative Verhoeff-van Gieson staining showing elastin degradation–grading keys (4 grades). Scale bar, 50 µm. B, Quantification of medial elastin degradation in WT (N=10), TWEAK KO (N=9) and Fn14 KO (N=9) mice. Values shown are mean±SE. C, Representative FMT ex-vivo images and (D) quantification of FMT aorta fluorescence from WT (N=6), TWEAK KO (N=6) and Fn14 KO (N=6) mice imaging datasets. Imaging was performed 24 hours following MMPSense-680 injection. Values shown are mean±SE. E, Gelatin gel zymography to detect MMP activities in tissue extract from AAA or thoracic aorta (T) from WT, TWEAK or Fn14 KO mice. AAA indicates abdominal aortic aneurysm; FMT, fluorescence molecular tomography; KO, knockout; MMP, matrix metalloproteinase; TWEAK, tumor necrosis factor-like weak inducer of apoptosis; WT, wild type.

Fig 5: TWEAK or Fn14 deletion reduced inflammatory cell content in abdominal aortic aneuryms. A, Representative staining and (B) quantification of positive staining of macrophages by MOMA-2 and neutrophils by Ly-6B.2 in AAA lesions from TWEAK KO (N=9), Fn14 KO (N=9) and WT (N=10) mice at 14 days postperfusion. Data are mean±SE. *P<0.05 vs WT. Negative controls were incubated with non-specific IgG. Scale bar, 50 µm. C, Representative staining and (D) quantification of CD3+ and CD31+ cells in AAA lesions from TWEAK KO (N=9), Fn14 KO (N=9) and WT (N=10) mice at 14 days postperfusion. Data are mean±SE. *P<0.05 vs WT. Negative controls were incubated with non-specific IgG. Scale bar, 50 µm. AAA indicates abdominal aortic aneurysm; KO, knockout; MOMA, monocyte macrophage antibody; TWEAK, tumor necrosis factor-like weak inducer of apoptosis; WT, wild type.