Inhibition of PCSK9 does not improve lipopolysaccharide-induced mortality in mice

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that targets LDL receptors (LDLRs) for degradation in liver. Blocking the interaction of PCSK9 with the LDLR potently reduces plasma LDL cholesterol levels and cardiovascular events. Recently, it has been suggested that inhibition of PCSK9 might also improve outcomes in mice and humans with sepsis, possibly by increasing LDLR-mediated clearance of endotoxins. Sepsis is a complication of a severe microbial infection that has shared pathways with lipid metabolism. Here, we tested whether anti-PCSK9 antibodies prevent death from lipopolysaccharide (LPS)-induced endotoxemia. Mice were administered PCSK9 antibodies prior to, or shortly after, injecting LPS. In both scenarios, the administration of PCSK9 antibodies did not alter endotoxemia-induced mortality. Afterward, we determined whether the complete absence of PCSK9 improved endotoxemia-induced mortality in mice with the germ-line deletion of Pcsk9 Similarly, PCSK9 knockout mice were not protected from LPS-induced death. To determine whether low LDLR expression increased LPS-induced mortality, Ldlr-/- mice and PCSK9 transgenic mice were studied after injection of LPS. Endotoxemia-induced mortality was not altered in either mouse model. In a human cohort, we observed no correlation between plasma inflammation markers with total cholesterol levels, LDL cholesterol, and PCSK9. Combined, our data demonstrate that PCSK9 inhibition provides no protection from LPS-induced mortality in mice.

Keywords: LDL; PCSK9; cholesterol; endotoxemia; lipopolysaccharide.

Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Figures

Fig. 1.

Administration of an anti-PCSK9 antibody does not reduce LPS-induced mortality. Survival curves of C57Bl/6J wild-type mice injected with control antibody (REGN1932, 10 mg/kg, sc) or alirocumab (10 mg/kg, sc) after LPS administration (n = 8 per group). Antibodies are injected 2 h after LPS inoculation: 7.5 mg/kg, ip (A), and 15 mg/kg, ip (B) at time 0. Mice were monitored hourly for 72 h and time of death recorded. C: Plasma inflammation markers in C57Bl/6J wild-type mice (n = 8 per group) treated with control antibody (REGN1932, 10 mg/kg, sc) or alirocumab (10 mg/kg, sc) before and 6 h after LPS (7.5 or 15 mg/kg, ip). All values represent means ± SEM. ALI, alirocumab; NS, no significant difference.

Fig. 2.

Alirocumab administered at various times does not change LPS-induced mortality. A: Plasma cholesterol levels of C57Bl/6J wild-type mice (n = 10 per group) treated with control antibody (REGN1932, 50 mg/kg, iv) or alirocumab (10 or 50 mg/kg, iv) before and 6 h after LPS (20 mg/kg, ip). Both doses of alirocumab were injected 1 or 2 h after LPS inoculation. All values represent means ± SEM. *P < 0.05. B: Survival curves of C57Bl/6J wild-type mice (n = 10 per group) injected with control antibody (REGN1932, 50 mg/kg, iv) or alirocumab (10 or 50 mg/kg, iv) 1 or 2 h after LPS (20 mg/kg, ip, time 0). Mice were monitored hourly for 72 h, and time of death was recorded.

Fig. 3.

Pretreatment with an anti-PCSK9 antibody does not protect mice from LPS-induced death. A: Plasma cholesterol levels 48 h after administration of a control antibody (REGN1932, 15 mg/kg, sc) or alirocumab (15 or 50 mg/kg, sc) injected into C57Bl/6J mice (n = 10 per group). Values represent means ± SEM. **P < 0.01. B: Survival curves of C57Bl/6J mice injected with LPS (20 mg/kg, ip) 48 h after the administration of the control antibody (REGN1932, 15 mg/kg, sc) or alirocumab (15 or 50 mg/kg, sc) (n = 10 per group). Mice are checked hourly for 72 h and time of death recorded. C: Plasma cholesterol levels of C57Bl/6J mice injected with control antibody (REGN1932, 10 mg/kg, sc) or alirocumab (10 mg/kg, sc) every week for 4 weeks (n = 12 per group). All values represent means ± SEM. *P < 0.05. D: Survival curves of C57Bl/6J mice injected with control antibody (REGN1932, 10 mg/kg, sc) or alirocumab (10 mg/kg, sc). LPS (20 mg/kg, ip) was injected (time 0) 48 h after the last antibody injection. Mice were monitored hourly for 72 h, and time of death was recorded.

Fig. 4.

PCSK9 antibody directed against mouse PCSK9 fails to improve LPS-induced mortality. A: Plasma cholesterol levels in C57Bl/6J mice (n = 11 per group) 48 h after control antibody (IgG1 MAB002, R&D Systems; 100 µg/mouse, sc) or anti-PCSK9 antibody (71207, BPS Bioscience; 100 µg/mouse, sc) injection. All values represent means ± SEM. ***P < 0.001. B: Survival curves in C57Bl/6J mice (n = 11 per group) injected with control antibody (IgG1 MAB002, R&D Systems; 100 µg/mouse, sc) or anti-PCSK9 antibody (71207, BPS Bioscience, 100 µg/mouse, sc). Antibodies were injected 48 and 1 h prior to LPS (20 mg/kg, ip, time 0). Mice were checked hourly for 72 h, and time of death was recorded. C: Survival curves of C57Bl/6J mice (n = 12 per group) injected with control antibody (IgG1 MAB002, R&D Systems) or anti-PCSK9 antibody (71207, BPS Bioscience; 100 µg/d/mouse, sc, 6 h after LPS (20 mg/kg, ip, time 0). Mice were monitored hourly for 72 h, and time of death was recorded.

Fig. 5.

Altering LDLR levels does not change LPS-induced mortality. A, C, and E: Plasma cholesterol levels of C57Bl/6J, Pcsk9+/+, Pcsk9−/−, Ldlr−/−, and Tg-hPCSK9 mice before LPS injection (n = 10 per group). All values represent means ± SEM. ***P < 0.001. B, D, and F: Survival curves of C57Bl/6J, Pcsk9+/+, Pcsk9−/−, Ldlr−/−, and Tg-hPCSK9 mice (n = 10 per group) after LPS injection (20 mg/kg, ip, time 0). Mice were monitored hourly for 72 h, and time of death was recorded.

Fig. 6.

No correlation between plasma inflammation markers and PCSK9 LOF mutations in humans. Concentrations of plasma PCSK9 (A) and cytokine levels (B) in subjects with PCSK9 LOF mutations (n = 28; six Y142X, four C679X, one R97DEL, one Y142X/R97DEL, and 16 with no PCSK9 mutation). All values represent means ± SEM. NM, no mutation.