Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Effect of LPS and malvidin on mitochondrial membrane potential of RAW 264.7 macrophages.

<p>Cells were pretreated or not with malvidin for 30 min and exposed or not to LPS for 1h. Medium was replaced to fresh one without any agents and containing 1 µg/ml JC-1 membrane potential-sensitive fluorescent dye for 15 min. Green and red fluorescence images of the same field were acquired using a fluorescent microscope. Representative merged images of three independent experiments are presented. Con: control; Mv: malvidin.</p

Effect of malvidin on LPS induced nuclear translocation and DNA binding of of NFκB.

<p>RAW 264.7 macrophages were treated for 1h as indicated, then nuclei were isolated and NFκB was extracted by using magnetic beads baited with oligonucleotides of the NFκB binding consesus sequence. Total (phosphorylated and unphosphorylated) NFκB (t-p65) as well as the phoshorylated form of its p65 subunit (p-p65) was detected by immunoblotting in the samples eluted from the beads. Histon H1 from the isolated nuclei was used as loading control. Representative blots (A) and densitometric evaluations (B,C) of three independent experiments are shown. Pixel densities were normalised to that of the histon H1. Values are given as means ± SEM. * p<0.05, ** p<0.01, *** p<0.001 compared to untreated control, ^### p<0.001 compared to LPS alone. a.u.: arbitrary units.</p

Effect of malvidin, kinase inhibitors and NAC on LPS induced nuclear translocation and DNA binding of of NFκB.

<p>RAW 264.7 macrophages were treated for 1h as indicated, then nuclei were isolated and NFκB was extracted using magnetic beads baited with oligonucleotides of NFκB binding consesus sequence. Total (phosphorylated and unphosphorylated) NFκB (t-p65) was detected by immunoblotting in the samples eluted from the beads. Histon H1 from the isolated nuclei was used as loading control. Representative blots (A) and densitometric evaluations (B) of 3 independent experiments are shown. Pixel densities were normalized to histon H1. Values are given as means ± SEM. * p<0.05, ** p<0.01, *** p<0.001 compared to untreated control, ^# p<0.05, ^## p<0.01, ^### p<0.001 compared to LPS alone. a.u.: arbitrary units; SB203580: p38 MAPK inhibitor; PD98059: ERK inhibitor; NAC: N-acetyl cysteine.</p

Effect of malvidin on LPS induced activation of Akt1 in RAW 264.7 macrophages.

<p>Steady state level of total (phosphorylated and unphosphorylated) Akt1 (t-Akt) as well as phosphorylation of Akt1 and its down-stream target GSK-3β was detected by immunoblotting from whole cell lysate after treating the cells as indicated for 1h. Actin was used as loading control. Representative blots (A) and densitometric evaluations (B–C) of 3 independent experiments are shown. Pixel densities were normalized to actin. Values are given as means ± SEM. *** p<0.001 compared to untreated control, ^### p<0.001 compared to LPS alone.</p

Effect of malvidin on LPS induced activation of NFκB in RAW 264.7 macrophages.

<p>Cells were pretreated with 0–100 µM malvidin (black bars in B) or 0–100 µM trans-resveratrol (gray bars in B) for 30 min as indicated. Activation of NF-κB was assessed by a luciferase (A) or an alkaline phosphatase (B) reporter assay after 1 µg/mL LPS exposion for 24 h. Values are given as means ± SEM of 4 independent experiments running in 3 parallels. ** p<0.01, *** p<0.001 compared to untreated control, ^# p<0.05, ^## p<0.01, ^### p<0.001 compared to LPS alone. a.u.: arbitrary units.</p

Effect of LPS and malvidin on MKP-1 expression in LPS treated RAW 264.7 macrophages.

<p>Effect of LPS and malvidin on steady state MKP-1 protein level was assessed by immunoblotting from whole cell lysate after treating the cells as indicated for 1h. Actin was used as a loading control. Representative blots (A) and densitometric evaluations (B) of 3 independent experiments are shown. Pixel densities were normalized to that of the actin. MKP-1 mRNA expression (C) was determined in another aliquot of cells treated as above using Q-RT-PCR analysis. β-Actin was used as a housekeeping control gene. Specific primer sequences and PCR conditions are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065355#s2" target="_blank">Materials and Methods</a>. Values are given as means ± SEM. * p<0.05, *** p<0.001 compared to untreated control, ^## p<0.01 ^### p<0.001 compared to LPS alone.</p