PCSK9 inhibition fails to alter hepatic LDLR, circulating cholesterol, and atherosclerosis in the absence of ApoE

Abstract

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15-30% lower circulating LDL-C and a disproportionately lower risk (47-88%) of experiencing a cardiovascular event. Here, we utilized pcsk9(-/-) mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9(-/-) mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (-45%) and TGs (-36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (-91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.

Keywords: anti-proprotein convertase subtilisin/kexin type 9 antibody; apolipoprotein E; low density lipoprotein receptor; proprotein convertase subtilisin/kexin type 9.

Fig. 1.

Minimal effect of deleting PCSK9 on circulating lipids or atherosclerosis in LDLR-deficient mice. A: Plasma PCSK9 levels are higher in ldlr^−/− mice compared with C57Bl/6 mice, consistent with the LDLR being a key clearance mechanism for PCSK9 (n = 9, ldlr^−/−; n = 12, C57Bl/6). The pcsk9^−/−/ldlr^−/− mice exhibit a slight decrease in TC (B) but not TGs (C) relative to ldlr^−/− mice when fed a WTD (n = 41, ldlr^−/−; n = 43, pcsk9^−/−/ldlr^−/−). D: The aortic sinus was sectioned and stained with VVG to measure lesion area (blue) and Mac-2 to monitor macrophage content (red). D–F: No difference in atherosclerosis development or macrophage accumulation was observed in the aortic sinus for ldlr^−/− mice relative to pcsk9^−/−/ldlr^−/− mice (n = 25 per group). Data represented as the means (bars) ± SD (*P < 0.05, ***P < 0.001, as compared with ldlr^−/−, two-tailed t-test, unpaired).

Fig. 2.

No effect of deleting PCSK9 on circulating lipids or atherosclerosis in APOE-deficient mice. A: Comparable levels of PCSK9 are observed in apoe^−/− mice compared with C57Bl/6 mice (n = 12, apoe^−/−; n = 12, C57Bl/6). No significant reduction in circulating TC (B) and TG (C) levels was observed in pcsk9^−/−/apoe^−/− mice relative to apoe^−/− mice (n = 9 (8 weeks), n = 21 (24 weeks) for pcsk9^−/−/apoe^−/−; n = 11 (8 weeks), n = 15 (24 weeks) for apoe^−/−). D–F: The aortic sinus was sectioned and stained with VVG to measure lesion area (blue) and Mac-2 to monitor macrophage content (red), and consistent with these observations, no difference in atherosclerosis development or macrophage accumulation was observed in the aortic sinus for apoe^−/− mice relative to pcsk9^−/−/apoe^−/− mice (n = 18, pcsk9^−/−/apoe^−/−; n = 20, apoe^−/−). Data represented as the means (bars) ± SD, two-tailed t-test, unpaired, as compared with apoe^−/−.

Fig. 3.

Anti-PCSK9 antibody treatment reduces TC and TG levels in APOE*3Leiden.CETP mice but not apoe^−/− mice. No significant reduction in TC (A) and only a slight but significant reduction in TGs 5 days posttreatment (B) are observed for anti-PCSK9 antibody-treated apoe^−/− mice relative to control antibody-treated apoe^−/− mice [10 mg/kg (sc) day 0, n = 5 per group]. This contrasts results with APOE*3Leiden.CETP mice, where anti-PCSK9 antibody treatment resulted in a significant decrease in TC (C) and TGs (D) (10 mg/kg (sc) day 0, n = 8 per group). Data represented as the means (bars) ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001, as compared with control, two-way ANOVA, Sidak posttest.

Fig. 4.

Anti-PCSK9 antibody treatment reduces atherosclerosis in APOE*3Leiden.CETP mice. Plasma PCSK9 levels (A) in APOE*3Leiden.CETP mice were determined on chow diet and WTD, as well as two weeks after a single injection with anti-PSCK9 antibody (10 mg/kg, sc) in mice fed WTD. Data represented as the means (bars) ± SD (n = 8 per group). *P < 0.01 versus chow; #P < 0.05 versus WD, one-way ANOVA, Tukey posttest. To assess the effect on atherosclerosis, control or anti-PCSK9 antibody was injected sc every 10 days for 14 weeks in APOE*3Leiden.CETP mice. Plasma TC (B) and TGs (C) were measured at 3 and 10 days postinjection in the first and twelfth week of treatment. Data represented as means (bars) ± SD (n = 15 per group). ***P < 0.001 versus control antibody. D: Fast protein liquid chromatography fractionation of pooled plasma samples are shown from week 8. Atherosclerosis development was determined in the aortic sinus of APOE*3Leiden.CETP mice. E–F: Representative pictures of control antibody- and anti-PCSK9 antibody-treated mice are shown. The total lesion area per cross-section (G) was measured and lesion severity (H) was determined. Data represented as means (bars) ± SD (n = 15 per group). ***P < 0.001 versus control antibody.