miR33 inhibition overcomes deleterious effects of diabetes mellitus on atherosclerosis plaque regression in mice

Abstract

Rationale: Diabetes mellitus increases cardiovascular disease risk in humans and remains elevated despite cholesterol-lowering therapy with statins. Consistent with this, in mouse models, diabetes mellitus impairs atherosclerosis plaque regression after aggressive cholesterol lowering. MicroRNA 33 (miR33) is a key negative regulator of the reverse cholesterol transport factors, ATP-binding cassette transporter A1 and high-density lipoprotein, which suggested that its inhibition may overcome this impairment.

Objective: To assess the effects of miR33 inhibition on atherosclerosis regression in diabetic mice.

Methods and results: Reversa mice, which are deficient in the low-density lipoprotein receptor and in which hypercholesterolemia is reversed by conditional inactivation of the microsomal triglyceride transfer protein gene, were placed on an atherogenic diet for 16 weeks, then either made diabetic by streptozotocin injection or kept normoglycemic. Lipid-lowering was induced by microsomal triglyceride transfer protein gene inactivation, and mice were treated with anti-miR33 or control oligonucleotides. Although regression was impaired in diabetic mice treated with control oligonucleotides, anti-miR33 treatment decreased plaque macrophage content and inflammatory gene expression in these mice. The decreased macrophage content in anti-miR33 treated diabetic mice was associated with a blunting of hyperglycemia-induced monocytosis and reduced monocyte recruitment to the plaque, which was traced to an inhibition of the proliferation of bone marrow monocyte precursors associated with the upregulation of their Abca1.

Conclusions: miR33 inhibition overcomes deleterious effects of diabetes mellitus in atherosclerosis regression in mice, which suggests a therapeutic strategy in diabetic patients, who remain at elevated cardiovascular disease risk, despite plasma cholesterol lowering.

Keywords: atherosclerosis; diabetes mellitus; high-density lipoprotein; microRNA; regression.

Figure 1. α-miR33 treatment derepresses its target genes in the liver of Reversa mice

(A) Experimental design: Reversa mice were placed on a western diet for 16 weeks. At 15 weeks, the mice received 5 injections of citrate (control) or 50mg/kg streptozotocin (diabetic). At 16 weeks, all mice were switched to a chow diet and received polyinosinic polycytidylic RNA (pIpC) 15 mg/kg every other day for a total of four injections to initiate lipid lowering. Mice then received weekly (s.c) injections of either control anti-miR or anti-miR33 2’F/MOE oligonucleotides (10mg/kg) for 4 weeks, prior to sacrifice (i.e 4 weeks post the final pIpC injection). Hepatic expression of (B) Abca1, (C) Abcg1, (D) Cpt1 and (E) Hmgcr. # p≤0.05 vs. con α-miR normoglycemic; * p≤0.05, *** p≤0.001 vs. con α-miR diabetic.

Figure 2. α-miR33 treatment restores regression in diabetic mice

Aortic roots from baseline and the regression groups were sectioned, fixed and stained for (A) CD68 and (B) collagen. Representative pictures of (A) CD68 immunostaining (magnification ×20) and (B) picrosirius red staining (under white and polarized light) of collagen (magnification ×10) are shown for each group. The areas of the plaques occupied by CD68+ cells and collagen (the latter as detected by polarized light) were quantified by Image Pro Plus Software and displayed in the graphs. Results are expressed as the percentage of plaque area. ^{^} p≤0.05 vs. baseline, ^# p≤0.05, ^### p≤0.001 vs. con α-miR normoglycemic; *** p≤0.001 vs. α-miR33 diabetic.

Figure 3. α-miR33 treatment affects monocyte trafficking to the plaque

Experimental design for monocyte trafficking. (A) Reversa mice were injected with fluorescent latex beads either 24h prior to harvesting (week 21) for the recruitment protocol or at week 14 of western diet for the egress protocol. (B) To assess monocyte recruitment mice were injected with fluorescent latex beads 24h prior to sacrifice, and beads counted in the aortic root. Data are expressed as mean ±SEM (n≥6 per group). ^## p≤0.01 vs. con α-miR normoglycemic ** p≤0.01 vs. con α-miR diabetic. (C) To assess macrophage retention mice were injected with fluorescent latex beads at week 14, and the number of beads remaining at sacrifice used to assess macrophage retention (baseline was established 1 week post bead injection). Data are expressed as mean ±SEM (n≥6 per group). ^{^^^} p≤0.001 vs. baseline. (D) Quantitative analysis of TUNEL⁺DAPI⁺ cells per section in the aortic root, after regression. Results are expressed as the percentage of total cells per plaque ±SEM (n=4 per group). (E) Quantitative analysis of necrotic core area as a percentage of total plaque area ±SEM (n≥6 per group) ^# p≤0.5 vs. con α-miR normoglycemic * p≤0.01 vs. con α-miR diabetic.

Figure 4. α-miR33 treatment decreases monocytosis in diabetic mice

Total monocytes and neutrophils were analyzed by flow cytometry in the blood at 16 weeks of diet (baseline) or after treatment. (A) Representative flow cytometry plots. Quantification of (B) monocytes and (C) neutrophils. Data are expressed as mean ±SEM (n≥10 per group). ^{^^}p≤0.01, *** p≤0.001 vs. baseline.

Figure 5. α-miR33 treatment decreases bone marrow progenitors in diabetic mice

C57Bl6/J mice were injected with citrate (normoglycemic) or 50mg/kg streptozotocin (diabetic). Mice were then injected 4 times with control α-miR or α-miR33 2’F/MOE oligonucleotides (10mg/kg). (A) Plasma blood glucose and (B) HDL-C 4 weeks post treatment. (C) Hematopoietic stem and progenitor cell populations in the BM were determined by flow cytometry and expressed as a percentage of cells in the BM. (D) Hematopoietic stem and progenitor cell cycle (G₂M phase) was assessed by flow cytometry using DAPI. Data are expressed as mean ± SEM, n≥7/group. ^#p≤0.05, ^##p≤0.01 vs. con α-miR normoglycemic; * p≤0.05 con α-miR diabetic.

Figure 6. α-miR33 treatment restores Abca1 expression in bone marrow progenitors of diabetic mice

C57Bl6/J mice were injected with citrate (normoglycemic) or 50mg/kg streptozotocin (diabetic). Mice were then injected 4 times with control α-miR or α-miR33 2’F/MOE oligonucleotides (10mg/kg). Gene expression for Abca1 and Abcg1 was assessed by qPCR in the different bone marrow progenitor subsets (A) LSK, (B) CMP and (C) GMP, obtained by cell sorting. Data are expressed as mean ± SEM, n≥7/group. ^#p≤0.05, ^##p≤0.01 vs. con α-miR normoglycemic ;* p≤0.05 vs. con α-miR diabetic.