Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides

Abstract

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention. MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3-5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels and providing protection against atherosclerosis; however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk.

Figure 1. Silencing of miR-33a/b in non-human primates

(a) Experimental outline of anti-miR33 or mismatch control oligonucleotide treatment in African green monkeys (n=6/group). (b) Serum transaminase (AST, ALT), bilirubin and creatinine levels. (c) Hepatic gene expression in anti-miR treated monkeys fed a chow diet (4 weeks), or a high carbohydrate, moderate cholesterol diet (12 weeks). (d) Quantitation of miR-33a and b levels in anti-miR treated monkeys. (e) Western blot for hepatic ABCA1, CPT1 and CROT following 12 weeks of anti-miR treatment. (f) Expression of hepatic SREBP1 mRNA and its downstream genes, and (g) SREBP1 protein after 12 weeks of anti-miR treatment. (h) Hepatic PRKAA1 and SIRT6 mRNA after 12 weeks of anti-miR treatment. Data are the mean ± SEM. *P ≤ 0.05.

Figure 2. Plasma cholesterol levels in control and anti-miR33 treated monkeys

Levels of plasma (a) total cholesterol, (b) HDL-cholesterol, (c) LDL-cholesterol and (d) VLDL-cholesterol in anti-miR treated monkeys. *P ≤ 0.05, † P ≤ 0.1. (e) Cholesterol content of FPLC fractionated lipoproteins.

Figure 3. Characterization of HDL

(a) Plasma apoAI and apoAII in anti-miR treated monkeys. *P ≤ 0.05. (b) HDL fractions (VL=very large, L=large, M=medium and S=small) analyzed by Western blot for apoE, apoAI and apoAII. (c) Macrophage cholesterol efflux to serum (2.5%) or PEG-isolated HDL from anti-miR treated monkeys. *P ≤ 0.05.

Figure 4. Triglyceride and VLDL Particle Analysis

Levels of plasma (a) total triglyceride, (b) VLDL-triglyceride, LDL-triglyceride or HDL-triglyceride from anti-miR treated monkeys. *P ≤ 0.05; † P ≤ 0.1. (c) Quantification of small, medium and large VLDL particle number by NMR spectroscopy. *P ≤ 0.05. (d) Western blot of apoE and apoB in VLDL fractions obtained from FPLC fractionation of plasma.