LDL-Cholesterol Reduction by ANGPTL3 Inhibition in Mice Is Dependent on Endothelial Lipase

Non-proprietary data that support the findings of this study are available from the corresponding author upon reasonable request.

Angiopoietin-like 3 (ANGPTL3), a protein secreted by the liver, is a regulator of lipoprotein metabolism¹. Humans carrying ANGPTL3 loss-of-function mutations have reduced plasma triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels. Antibody-mediated inhibition of ANGPTL3 reduces TG, LDL-C, and HDL-C in mice², monkeys², and humans³. ANGPTL3 inhibits lipoprotein lipase (LPL) and endothelial lipase (EL). The TG- and HDL-C-lowering effects of ANGPTL3 inhibition are due to increased LPL and EL activity, respectively²; however, the mechanism for lowering LDL-C remains a mystery. It appears to be independent of the LDL receptor (LDLR), as ANGPTL3 inhibition reduces LDL-C in Ldlr knockout (KO) mice⁴ and patients with homozygous familial hypercholesterolemia (hoFH)³.

We previously reported that hepatic overexpression of EL in hypercholesterolemic mice reduced non-HDL-C, LDL-C, and apoB levels⁵, suggesting that increased EL activity as a result of ANGPTL3 inhibition could contribute to the LDL-C reduction. In this report, we tested the dependence of LDL-C reduction upon Angptl3 silencing on EL by using mice lacking EL (EL-KO). Because wild-type (WT) and EL-KO mice have extremely low LDL-C levels, we raised LDL-C by using an siRNA to silence the hepatic Ldlr. Then we administered an Angptl3 siRNA or a control siRNA and compared the changes in plasma lipid levels in WT and EL-KO mice.

Fig 1A shows the study design. Two independent experiments were performed and were concordant; results are shown for the first experiment. Hepatic LDLR was undetectable by Western blot (Fig 1B). Angptl3 siRNA robustly reduced hepatic Angptl3 mRNA and plasma ANGPTL3 levels (Fig 1C). Following Ldlr siRNA injection, all mice had a significant increase in plasma cholesterol and non-HDL-C (Fig 1D,E). Injection of Angptl3 siRNA significantly decreased plasma TG levels to a similar extent in WT and EL-KO mice but decreased total cholesterol and HDL-C only in WT and not in EL-KO mice (Fig 1D). Importantly, Angptl3 silencing also significantly reduced plasma non-HDL-C, VLDL-C, and LDL-C in WT but not in EL-KO mice (Fig 1E). These results were confirmed in a separate independent experiment. Our data indicate that EL not only mediates the HDL-C lowering effect of ANGPTL3 inhibition but also plays a necessary role in mediating the LDLR-independent reduction in LDL-C.

Figure 1.

A) Study design: 8–10 week-old WT (n=10) and EL-KO (n=10) mice on chow diet were used for the study (n=5/treatment group). Fasting bleeds are marked with *. Two independent experiments of this design were performed. The mice and protocol used were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania. B) Hepatic LDLR protein: Primary antibodies: anti-LDLR (1:5,000, Abcam ab52818), anti-β-Actin (1:10,000 Sigma-Aldrich A5441). C) Hepatic Angptl3 expression: mRNA by qRT-PCR and plasma ANGPTL3 levels by ELISA. Data are presented as mean±SEM. Mann-Whitney test was performed to compare between treatment groups. D) Fasting plasma lipids: Measured by autoanalyzer. E) Non-HDL-C, VLDL-C, and LDL-C: Non-HDL-C = TC minus HDL-C. Pooled plasma was fractionated by FPLC and used to determine the % of individually measured non-HDL-C that was VLDL-C and LDL-C. P-values (bold) in Figure 1D and E show overall treatment effect as per repeated-measures two-way ANOVA. Significant comparisons by Sidak’s multiple comparisons test between treatment groups are shown at the indicated time points.

We previously showed that overexpression of EL in LDLR-KO mice increased phospholipase activity that accelerated LDL clearance from plasma⁵. Thus, increased EL activity in ANGPTL3 inhibition may accelerate LDL catabolism leading to decreased LDL-C. While kinetic studies in carriers of ANGPTL3 loss-of-function mutations showed increased LDL particle clearance¹, immunological inhibition of ANGPTL3 in WT mice did not⁴. Alternatively, EL may promote the clearance of LDL precursors, namely VLDL and intermediate-density lipoproteins, leading to reduced LDL production. Although it remains unclear precisely how EL mediates LDL-C lowering in ANGPTL3 inhibition, our study indicates that EL plays a crucial role in this process. Given the interest in ANGPTL3 inhibition as a therapeutic approach to reduce LDL-C, this insight into the mechanism of LDL reduction with ANGPTL3 inhibition is potentially important and suggests that variation in LDL-C reduction with ANGPTL3 inhibition could be due in part to variation in EL activity.