Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct;140(14):1170-1184.
doi: 10.1161/CIRCULATIONAHA.119.039476. Epub 2019 Sep 30.

Apolipoprotein AI) Promotes Atherosclerosis Regression in Diabetic Mice by Suppressing Myelopoiesis and Plaque Inflammation

Tessa J Barrett  1 , Emilie Distel  1 , Andrew J Murphy  2   3 , Jiyuan Hu  4 , Michael S Garshick  1 , Yoscar Ogando  1 , Jianhua Liu  5 , Tomas Vaisar  6 , Jay W Heinecke  6 , Jeffrey S Berger  7   8 , Ira J Goldberg  9 , Edward A Fisher  1   10
Affiliations

Apolipoprotein AI) Promotes Atherosclerosis Regression in Diabetic Mice by Suppressing Myelopoiesis and Plaque Inflammation

Tessa J Barrett et al. Circulation. 2019 Oct.

Abstract

Background: Despite robust cholesterol lowering, cardiovascular disease risk remains increased in patients with diabetes mellitus. Consistent with this, diabetes mellitus impairs atherosclerosis regression after cholesterol lowering in humans and mice. In mice, this is attributed in part to hyperglycemia-induced monocytosis, which increases monocyte entry into plaques despite cholesterol lowering. In addition, diabetes mellitus skews plaque macrophages toward an atherogenic inflammatory M1 phenotype instead of toward the atherosclerosis-resolving M2 state typical with cholesterol lowering. Functional high-density lipoprotein (HDL), typically low in patients with diabetes mellitus, reduces monocyte precursor proliferation in murine bone marrow and has anti-inflammatory effects on human and murine macrophages. Our study aimed to test whether raising functional HDL levels in diabetic mice prevents monocytosis, reduces the quantity and inflammation of plaque macrophages, and enhances atherosclerosis regression after cholesterol lowering.

Methods: Aortic arches containing plaques developed in Ldlr-/- mice were transplanted into either wild-type, diabetic wild-type, or diabetic mice transgenic for human apolipoprotein AI, which have elevated functional HDL. Recipient mice all had low levels of low-density lipoprotein cholesterol to promote plaque regression. After 2 weeks, plaques in recipient mouse aortic grafts were examined.

Results: Diabetic wild-type mice had impaired atherosclerosis regression, which was normalized by raising HDL levels. This benefit was linked to suppressed hyperglycemia-driven myelopoiesis, monocytosis, and neutrophilia. Increased HDL improved cholesterol efflux from bone marrow progenitors, suppressing their proliferation and monocyte and neutrophil production capacity. In addition to reducing circulating monocytes available for recruitment into plaques, in the diabetic milieu, HDL suppressed the general recruitability of monocytes to inflammatory sites and promoted plaque macrophage polarization to the M2, atherosclerosis-resolving state. There was also a decrease in plaque neutrophil extracellular traps, which are atherogenic and increased by diabetes mellitus.

Conclusions: Raising apolipoprotein AI and functional levels of HDL promotes multiple favorable changes in the production of monocytes and neutrophils and in the inflammatory environment of atherosclerotic plaques of diabetic mice after cholesterol lowering and may represent a novel approach to reduce cardiovascular disease risk in people with diabetes mellitus.

Keywords: apolipoproteins; arteriosclerosis; diabetes mellitus; leukocytosis; lipoproteins, HDL; macrophages; myelopoiesis.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. Hyperglycemia correlates with elevated white blood cell count and reduced HDL-C in humans and mice with diabetes.
(A) White blood cell count and (B) HDL-C correlates with HbA1c in humans (n=146, n=102). (C) White blood cell count and (D) HDL-C correlates with blood glucose in diabetic mice (n=22). Pearson’s correlation coefficient (ρ) and p values were assessed in R. Black triangles and lines represent observations and the fitted regression lines.
Figure 2.
Figure 2.. Elevating HDL overcomes hyperglycemia-mediated leukocytosis.
(A) HDL and (B) human apoA-I levels are raised in apoA-I Tg mice compared to wild-type (WT) controls (n=10). (C-D) HDL-P spectra of WT control mice and mice overexpressing apoA-I/HDL (representative images). (E) White blood cell counts in WT, diabetic WT, apoA-I Tg, and diabetic apoA-I Tg mice (n=8–10/grp). (F) Representative flow cytometry plots of blood leukocyte subsets from WT, diabetic WT, apoA-I Tg, and diabetic apoA-I Tg mice. Red box depicts monocyte population. (G) Quantification of monocytes, and monocyte subsets and (H) neutrophils (n=8–10/grp). Neutrophil (I) CD11b and (J) ROS in WT, diabetic WT, apoA-I Tg, and diabetic apoA-I Tg mice (n=8–10/grp). In panel A, data are expressed as mean ± SEM. * p<0.0001 determined by a t-test (two-tailed). Data are expressed as mean ± SEM. * p<0.01 compared to WT control group or ^ p<0.01 compared to WT STZ group as determined via 2-way ANOVA and Tukey’s post-hoc test.
Figure 3.
Figure 3.. Raising apoA-I/HDL in diabetic mice suppresses hyperglycemia-driven myeloproliferation by promoting cholesterol efflux and suppressing neutrophil S100A8/A9 production.
(A) Representative flow cytometry of monocyte and neutrophil precursors, common myeloid progenitors (CMPs), and granulocyte macrophage progenitors (GMPs). (B) Quantification of CMPs and GMPs in the bone marrow in WT, diabetic WT, apoA-I Tg, and diabetic apoA-I Tg mice (n=7–9/grp). Abca1 and Abcg1 expression in (C) CMP and (D) GMP populations within the bone marrow (n=7–9/grp). (E) Cholesterol efflux capacity of monocyte progenitors (CMPs & GMPs) for WT, diabetic WT, apoA-I Tg and diabetic apoA-I Tg mice (n=9 mice/grp). (F) CMP and GMP cell cycle analyses in the bone marrow in WT, diabetic WT, apoA-I Tg, and diabetic apoA-I Tg mice (n=7–9/grp). (G) s1008, s100a9 and rage expression in neutrophils isolated from WT, diabetic WT, apoA-I Tg, and diabetic apoA-I Tg mice (n=6/grp). (H) Circulating S100A8/A9 plasma protein levels, (I) bone marrow rage expression, (J) CMP RAGE surface expression (n=7–9/grp). Data are expressed as mean ± SEM. * p<0.01 compared to WT control group or ^ p<0.01 compared to WT STZ group as determined via 2-way ANOVA and Tukey’s post-hoc test in panel (B)-(D), (F)-(J). * p<0.05 compared to the control group or ^ p<0.05 compared to the diabetic BSA group for WT and ApoA-I Tg mice respectively as determined via 1-way ANOVA and Tukey’s post-hoc test in panel (E).
Figure 4.
Figure 4.. HDL-C is negatively associated with leukocyte subsets and inflammation.
(A) Neutrophil, and (B) monocyte counts stratified by HDL-C (< 40 mg/dL n=127, >40 mg/dL n=157) and (C) CD14+CD16+ monocyte populations as stratified by HDL-C (< 40 mg/dL n=24, >40 mg/dL n=28). (D) Plasma S100A9 concentration as determined via ELISA (< 40 mg/dL n=8, >40 mg/dL n=6). Subjects are derived from the patient cohort present in Table 1. Data are expressed as mean ± SEM. * p<0.01, ** p<0.005 as determined via Mann-Whitney U test.
Figure 5.
Figure 5.. Raising apoA-I/HDL promotes atherosclerosis regression in diabetic mice.
(A) Atherosclerosis regression protocol. (B) Total cholesterol, (C) blood glucose, (D) white blood cell counts (E) monocyte counts in baseline, WT, diabetic WT and diabetic apoA-I Tg mice (n= 7–11 mice/grp). (F) Representative images and (G) quantification of plaque CD68 staining. (H) Representative images and (I) quantification of plaque mannose receptor expression. (J) Representative images and (K) quantification of plaque collagen content. (L) Representative images and (M) quantification of plaque Ki67 staining (n=7–11 mice/grp). Data are expressed as mean ± SEM. *** p<0.001, ** p<0.01, * p<0.05 compared to the baseline group as determined via 1-way ANOVA and Tukey’s post-hoc test in panels (B), (C), (D), (E) and (M). * p<0.01, compared to the WT STZ group as determined via 1-way ANOVA and Tukey’s post-hoc test in panels (G), (I), and (K).
Figure 6.
Figure 6.. Increased circulating levels of apoA-I/HDL suppresses monocyte recruitment to diabetic plaques, and to sites of inflammation.
(A) Monocyte and macrophage plaque trafficking protocol. Representative image of (B) macrophage retention and (C) quantification in regressing atherosclerotic plaques (n=6–8 mice/grp). Representative image of (D) monocyte recruitment and (E) quantification in regressing atherosclerotic plaques (n=6–8 mice/grp). Data are expressed as mean ± SEM. * p <0.01, compared to the baseline group as determined via 1-way ANOVA and Tukey’s post-hoc test. (F) Representative flow cytometry plots of monocyte and neutrophil recruitment to the peritoneal cavity following zymozan injection. (G) Quantification of recruited leukocytes, monocytes and neutrophils to the peritoneal cavity following a zymozan challenge in wild-type (WT) and apoA-I Tg mice (n=3–7 mice/grp). Data are expressed as mean ± SEM. * p <0.01, compared to WT group, and ^ p <0.01 compared to zymozan injected WT group as determined via 2-way ANOVA and Tukey’s post-hoc test. (H) Monocyte and neutrophil chemotaxis to CCL5 following pre-treatment with apoA-I (40 ug.mL). Data are expressed as mean ± SEM. * p<0.01, compared to the no chemokine control, and ^ p<0.01, compared to CCL5-stimulated cells as determined via 1-way ANOVA and Tukey’s post-hoc test.
Figure 7.
Figure 7.. Plaque neutrophil extracellular traps are elevated in regressing plaques from diabetic mice, and are reduced following raising apoA-I/HDL.
(A) Representative images of plaque neutrophil extracellular traps (NETs), as determined by citrullinated histone h3 (H3C), neutrophil (Ly6G), and myeloperoxidase (MPO) costaining. (B) Quantification of plaque NET content (n=6–10/grp). Data are expressed as mean ± SEM. * p<0.01, compared to the baseline group as determined via 1-way ANOVA and Tukey’s post-hoc test.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Haffner SM, Lehto S, Ronnemaa T, Pyorala K and Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. New Eng J Med. 1998;339:229–234. - PubMed
    1. Cholesterol Treatment Trialists C, Baigent C, Blackwell L, Emberson J, Holland LE, Reith C, Bhala N, Peto R, Barnes EH, Keech A, et al. Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet. 2010;376:1670–1681. - PMC - PubMed
    1. Hiro T, Kimura T, Morimoto T, Miyauchi K, Nakagawa Y, Yamagishi M, Ozaki Y, Kimura K, Saito S, Yamaguchi T, et al. Diabetes mellitus is a major negative determinant of coronary plaque regression during statin therapy in patients with acute coronary syndrome--serial intravascular ultrasound observations from the Japan Assessment of Pitavastatin and Atorvastatin in Acute Coronary Syndrome Trial (the JAPAN-ACS Trial). Circulation journal : official journal of the Japanese Circulation Society. 2010;74:1165–1174. - PubMed
    1. Distel E, Barrett TJ, Chung K, Girgis NM, Parathath S, Essau CC, Murphy AJ, Moore KJ and Fisher EA. miR33 inhibition overcomes deleterious effects of diabetes mellitus on atherosclerosis plaque regression in mice. Circ Res. 2014;115:759–769. - PMC - PubMed
    1. Gaudreault N, Kumar N, Olivas VR, Eberle D, Stephens K and Raffai RL. Hyperglycemia impairs atherosclerosis regression in mice. Am J Pathol. 2013;183:1981–1992. - PMC - PubMed

Publication types

MeSH terms