WISP1 alleviates lipid deposition in macrophages via the PPARγ/CD36 pathway in the plaque formation of atherosclerosis

Abstract

Lipid deposition in macrophages plays an important role in atherosclerosis. The WNT1-inducible signalling pathway protein 1(WISP1) can promote proliferation and migration of smooth muscle cells. Its expression is up-regulated in obesity, which is associated with atherosclerosis, but the effect of WISP1 on atherosclerosis remains unclear. Thus, the objective of our study was to elucidate the role of WISP and its mechanism of action in atherosclerosis via in vivo and in vitro experiments. In our experiment, ApoE-/- mice were divided into 5 groups: control, high-fat diet (HFD), null lentivirus (HFD + NC), lentivirus WISP1 (HFD + IvWISP1) and WISP1-shRNA (HFD + shWISP1). Oil Red O staining, immunofluorescence and immunohistochemistry of the aortic sinuses were conducted. Macrophages (RAW264.7 cell lines and peritoneal macrophages) were stimulated with 50 μg/mL oxidized low-density lipoprotein (ox-LDL); then, the reactive oxygen species (ROS) level was measured. Oil Red O staining and Dil-ox-LDL (ox-LDL with Dil dye) uptake measurements were used to test lipid deposition of peritoneal macrophages. WISP1, CD36, SR-A and PPARγ expression levels were measured via Western blotting and ELISA. The results showed that HFD mice had increased WISP1, CD36 and SR-A levels. The plaque lesion area increased when WISP1 was down-regulated, and lipid uptake and foam cell formation were inhibited when WISP1 was up-regulated. Treatment of RAW264.7 cell lines with ox-LDL increased WISP1 expression via activation of the Wnt5a/β-catenin pathway, whereas ROS inhibition reduced WISP1 expression. Moreover, WISP1 down-regulated CD36 and SR-A expression, and Oil Red O staining and Dil-ox-LDL uptake measurement showed that WISP1 down-regulated lipid deposition in macrophages. These results clearly demonstrate that WISP1 is activated by ox-LDL at high ROS levels and can alleviate lipid deposition in atherosclerosis through the PPARγ/CD36 pathway.

Keywords: CD36; PPRAγ; SR-A; WISP1; atherosclerosis; lipid deposition; macrophage.

Figure 1

Characteristics of ApoE‐/‐ mice. TC (A, total cholesterol), HDL‐C (B, high‐density lipoprotein cholesterol), LDL‐C (C, low‐density lipoprotein cholesterol), and WISP1 (D, WNT1‐inducible signalling pathway protein 1) levels in plasma, and weight (E) were measured after 12 weeks of a high‐fat diet (*P < .05; **P < .01; data = means ±SD). HFD: high‐fat diet; NC: null lentivirus; IvWISP1: lentivirus WISP1; shWISP1: WISP1‐shRNA

Figure 2

Down‐regulation of WISP1 promotes plaque formation, and WISP1 overexpression alleviates plaque formation in ApoE‐/‐ mice. (A) Representative image of haematoxylin and eosin–stained images of cross‐sections from the aortic sinus. (B) Representative image of Oil Red O staining of the aortic sinus (scale bar: 100 μm). (C) Representative image of Oil Red O staining of whole aortas (*P < .05, **P < .01, ***P < .001; data = means ±SD). HFD: high‐fat diet; NC: null lentivirus; IvWISP1: lentivirus WISP1; shWISP1: WISP1‐shRNA; WISP1: WNT1‐inducible signalling pathway protein 1

Figure 3

WISP1 leads to lipid deposition and recruitment of macrophages in ApoE‐/‐ mice. Immunohistochemical staining of WISP1 (A) and MOMA2 (B) from the aortic sinus (scale bar: 50 μm). (C) Immunofluorescent staining of CD36 in the aortic sinus (scale bar: 50 μm) (*P < .05, **P < .01, ***P < .001; data = means ±SD). HFD, high‐fat diet; NC, null lentivirus; IvWISP1, lentivirus WISP1; shWISP1, WISP1‐shRNA; WISP1, WNT1‐inducible signalling pathway protein 1

Figure 4

WISP1 reduces scavenger receptor (CD36 and SR‐A) levels in the arteries of ApoE‐/‐ mice. Western blot analysis of WISP1 (A), CD36 (B) and SR‐A (C). HFD can up‐regulate WISP1 via the Wnt5a/β‐catenin pathway. Western blot analysis of Wnt5a (D), β‐catenin (E) and WISP1 (F) (*P < .05, **P < .01, ***P < .001; data = means ±SD). HFD: high‐fat diet; NC: null lentivirus; IvWISP1: lentivirus WISP1; shWISP1: WISP1‐shRNA; WISP1: WNT1‐inducible signalling pathway protein

Figure 5

Ox‐LDL induced WISP1 expression through ROS in macrophages via the Wnt5a/β‐catenin pathway. (A) ROS levels were detected via immunofluorescence. (B) Western blot analysis of Wnt5a (B1), β‐catenin (B2) and WISP1 (B3) (*P < .05, **P < .01, ***P < .001; data = means ±SD). NAC: N‐acetylcysteine; ROS: reactive oxygen species; WISP1: WNT1‐inducible signalling pathway protein 1; ox‐LDL: oxidized low‐density lipoprotein

Figure 6

Down‐regulation of WISP1 can accelerate lipid deposition in macrophages. A: Western blot analysis of WISP1 (A1), PPARγ (A2), CD36 (A3) and SR‐A (A4) in macrophages. B: Representation Oil Red O staining of macrophage (scale bar: 25 μm, n = 7‐9). C: Wisp1 content in cell supernatant. D: Representative image of fluorescently labelled oxidized low‐density lipoprotein (Dil‐ox‐LDL) uptake in macrophages (scale bar: 25 μm, n = 5) (*P < .05, **P < .01, ***P < .001; data are means ± SD). NC: null lentivirus, IvWISP1: lentivirus WISP1, shWISP1: WISP1‐shRNA

Figure 7

Down‐regulation of WISP1 is involved in lipid deposition through PPARγ pathway. A: Western blot analysis of WISP1, PPARγ, CD36 and SR‐A in macrophages. B: Representation of Oil Red O staining of macrophage (scale bar: 25 μm) in macrophages (*P < .05, **P < .01, ***P < .001; data are means ± SD). shWISP1: WISP1‐shRNA, T0070907: a kind of PPARγ inhibitor. WISP1‐shRNA

Figure 8

Proposed schematic model (A) Ox‐LDL can depolarize mitochondrial membrane and trigger excessive reactive oxygen species (ROS). ROS can activate Wnt5a/β‐catenin pathway and then up‐regulate expression of WISP1. While ROS inhibition NAC can reduced WISP1 expression under the stimulation of ox‐LDL. (B) WISP1 expressed in the cytosol where it catalyses ubiquitination and subsequent degradation of PPARγ through direct interaction, consequently inhibiting its transcriptional activity. Inhibition of PPARγ can inhibit its downstream CD36. Besides, WISP1 can down‐regulate the expression of SR‐A. CD36 and SR‐A are crucial for lipid deposition