Figure 5.

KAL upregulates SIRT1 activity and fenofibrate upregulates KAL expression. (A) SIRT1 activity was assessed in the nuclear extract from IRA and SRA segments. Compared to the respective controls, the nuclear protein extract obtained from the CaPO₄ administered KS-Tg mice and rhKAL-administered AngII-induced ApoE^−/− mice showed increased SIRT1 activity (n = 8/group). (B) Relative SIRTI mRNA expression in AAA-neck (n = 6) and AAA-body (n = 12) tissues. Aortic tissues were collected in the RNAlater and total RNA isolated to perform quantitative real time PCR (QRT–PCR). (C) AAA-Body and AAA-neck tissues (n = 6) were used for isolating VSMCs using established protocol. AAA-VSMCs were collected in the RNA later and total RNA isolated to perform QRT-PCR to assess SIRT1 mRNA expression. (D,E) Incubation of VSMCs with increasing concentration of fenofibrate upregulated KAL protein expression. Cells were plated at 0.5 × 10⁶ cells in 500 µl DMEM + 5% FBS and incubated with fenofibrate (0–100 µM) over 24 h after which protein and mRNA was assayed (n = 6/group). Incubation of VSMCs with increasing concentration of fenofibrate (0–100 µM) dose-dependently stimulated (D) SERPINA4 protein and (E) SERPINA4 mRNA expression. Protein concentrations were assessed by Bradford method and SERPINA4 concentration levels were assessed in the cell culture supernatants by ELISA. QRT-PCR was performed on extracted total mRNA using SERPIN4A primers and normalised to GAPDH expression. (n = 6 replicates). (F) AAA-VSMCs (n = 6) were incubated with fenofibrate (100 µM) for 24 h. Total RNA was isolated from the cells SERPINA4 mRNA expression was assessed by QRT-PCR. Analysis performed by Mann Whitney U or Kruskal Wallis tests and statistical significance shown as *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH, glyceraldehyde 3 phosphate; IRA, infrarenal aorta; KS-Tg, kallistatin transgenic; rhKAL, recombinant human kallistatin; RFU, relative fluorescent unit; SERPINA4, serpin-A4; SIRT1, sirutuin-1; SRA, suprarenal aorta; VC, vehicle control; VSMC, vascular smooth muscle cell; WT, wild type.