Subclinical atherosclerosis and its progression are modulated by PLIN2 through a feed-forward loop between LXR and autophagy

Abstract

Background: Hyperlipidaemia is a major risk factor for cardiovascular disease, and atherosclerosis is the underlying cause of both myocardial infarction and stroke. We have previously shown that the Pro251 variant of perilipin-2 reduces plasma triglycerides and may therefore be beneficial to reduce atherosclerosis development.

Objective: We sought to delineate putative beneficial effects of the Pro251 variant of perlipin-2 on subclinical atherosclerosis and the mechanism by which it acts.

Methods: A pan-European cohort of high-risk individuals where carotid intima-media thickness has been assessed was adopted. Human primary monocyte-derived macrophages were prepared from whole blood from individuals recruited by perilipin-2 genotype or from buffy coats from the Karolinska University hospital blood central.

Results: The Pro251 variant of perilipin-2 is associated with decreased intima-media thickness at baseline and over 30 months of follow-up. Using human primary monocyte-derived macrophages from carriers of the beneficial Pro251 variant, we show that this variant increases autophagy activity, cholesterol efflux and a controlled inflammatory response. Through extensive mechanistic studies, we demonstrate that increase in autophagy activity is accompanied with an increase in liver-X-receptor (LXR) activity and that LXR and autophagy reciprocally activate each other in a feed-forward loop, regulated by CYP27A1 and 27OH-cholesterol.

Conclusions: For the first time, we show that perilipin-2 affects susceptibility to human atherosclerosis through activation of autophagy and stimulation of cholesterol efflux. We demonstrate that perilipin-2 modulates levels of the LXR ligand 27OH-cholesterol and initiates a feed-forward loop where LXR and autophagy reciprocally activate each other; the mechanism by which perilipin-2 exerts its beneficial effects on subclinical atherosclerosis.

Keywords: 27OH-cholesterol; PLIN2; atherosclerosis; autophagy; liver-X-receptor.

Figure 1

PLIN2 functionality has significant consequences on carotid plaque growth and macrophage infiltration. (a) The % core area and (b) CD68 staining using immunohistochemistry of 40 human carotid atherosclerotic plaques from the Carotid Plaque Imaging Project carrying either variant of PLIN2, matched for age, sex and diabetes status. Data are presented as median and IQR.

Figure 2

PLIN2 modulates the availability of 27‐HC and thereby also LXR activity. (a–c) PLIN2 expression by variant in PLIN2 as shown by Western blotting and qPCR. (d–e) Cholesterol ester and 27‐HC accumulation upon oxLDL challenge. Data are presented as median and IQR (b, d, e), and mean ± SEM (c). See also Fig. S1.

Figure 3

PLIN2 comprises a hub in cholesterol metabolism by connecting LXR activity and autophagy. (a) LXR target co‐expression by PLIN2 variant. (b) Cholesterol efflux without 27‐HC supplementation to the respective acceptor by protein variant of PLIN2. (c) Cholesterol efflux with 27‐HC supplementation to the respective acceptor by protein variant of PLIN2. (d) Autophagy activity, displayed as LC3 and p62 flux, by PLIN2 variant using Western blotting. (e, f) Densitometry of bands obtained from Western blotting. Autophagy blockade using bafilomycin A1 was applied as indicated. Data are presented as mean ± SEM (b), and median and IQR (d, e). **P < 0.01, ***P < 0.001, ****P < 0.0001. See also Fig. S2.

Figure 4

An in vitro system of HEK293 cells confirms that the functional PLIN2 protein variant modulates LXR activity. LXR was stimulated or inhibited using GW3965 and GSK2033, and autophagy was either stimulated or inhibited as indicated. (a) LXR luciferase activity in HEK293 cells carrying either variant of PLIN2. (b, c) Autophagy activity, displayed as LC3 flux, in response to LXR activation. (d) LXR Luciferase activity in response to rapamycin and 3‐methyladenine (3MA) treatment. Data are presented as mean ± SEM (a, d), and median and IQR (c). See also Fig. S2.

Figure 5

LXR and autophagy regulates their reciprocal activation in human primary monocyte‐derived macrophages. (a) mRNA expression of LXR targets SREBP1c and ABCA1, and PLIN2 upon manipulation of LXR activity using GW3965 and GSK2033. (b) Autophagy activity, displayed as LC3 flux, upon manipulation of LXR activity using GW3965 and GSK2033. (c, d) Gene expression and autophagy activity, displayed as LC3 flux, in monocyte‐derived macrophages induced by rapamycin stimulation. Data are presented as mean ± SEM.

Figure 6

27‐HC serves as a pivotal link in the crosstalk between LXR and autophagy in human primary monocyte‐derived macrophages. (a) 27‐HC measurements in response to oxLDL, rapamycin or 3‐methyladenine. (b) mRNA expression LXR targets SREBP1c, ABCA1 and CYP27A1 upon 27‐HC treatment. (c) Autophagy activity, displayed as LC3 flux, upon 27‐HC treatment. (d) mRNA expression LXR targets SREBP1c, ABCA1 and CYP27A1, upon CYP27A1 silencing. (e) Autophagy activity, displayed as LC3 flux, upon CYP27A1 silencing. Data are presented as mean ± SEM.