Increased inflammation, endoplasmic reticulum stress and oxidative stress in endothelial and macrophage cells exacerbate atherosclerosis in ApoCIII transgenic mice

Abstract

Background: Overexpression of apolipoprotein CIII (ApoCIII) leads to hypertriglyceridemia (HTG) which promotes atherosclerosis development. However, it remains unclear whether ApoCIII affects the atherosclerosis alone by promoting the inflammation and endoplasmic reticulum (ER) stress, or in combination with HTG.

Methods: Transgenic (ApoCIIItg) mouse models were used to investigate the atherogenic role of ApoCIII. Since endothelial cells and macrophages play crucial roles in atherosclerosis, we examined whether triglyceride-rich lipoproteins (TRLs), the major lipoproteins, in plasma of ApoCIIItg mice affect inflammation and ER stress levels in these cells. To further investigate the role of ApoCIII and triglyceride, we incubated HUVECs cells and peritoneal macrophages with TRLs with or without ApoCIII.

Results: Increased inflammation and ER stress were found in the aorta of ApoCIIItg mice. TRLs increased ER stress and oxidative stress in HUVECs and macrophages in a dose dependent. Moreover, TRLs together with ApoCIII could induce a higher inflammation level than TRLs alone in these cells.

Conclusions: Both TRLs and ApoCIII contribute to the progression of atherosclerosis, and the modulation of TRLs and ApoCIII may represent a novel therapeutic approach against HTG induced atherosclerosis.

Keywords: Apolipoprotein CIII; Endoplasmic reticulum stress; Endothelial cells; Inflammation; Macrophages; Oxidative stress; Triglyceride-rich lipoprotein.

Fig. 1

Increased inflammation, ER stress and oxidative stress in aorta of ApoCIIItgLDLR−/− mice comparing to LDLR−/− mice. (a) Representative images of Oil Red O (ORO) stained aortic roots and immunohistochemical staining of aortic sinus sections of Mac2, 4HNE and VCAM-1 expression in LDLR−/− and ApoCIIItgLDLR−/− mice. (b) Representative Western blot images of VCAM-1 and ICAM-1 protein expression in aortas of LDLR−/− and ApoCIIItgLDLR−/− mice and the protein quantification by densitometry (n = 4). (c) Representative Western blot images of GRP78 protein expression in aortas of LDLR−/− and ApoCIIItgLDLR−/− mice and the protein quantification by densitometry (n = 4). Values are expressed as mean ± SEM, *p < 0.05 **p < 0.01

Fig. 2

TRLs increase inflammation in HUVECs and macrophages dependent on ApoCIII. (a) MCP1 expression in HUVECs incubated with 100 μg/ml TRLs for 48 h (n = 4), **p < 0.01. (b) Western blot images (up) and the protein quantification (down) of VCAM-1 in HUVECs after the incubation with 20 μg/ml TRLs for 24 h and 1 μg/ml LPS for 6 h before the analysis (n = 4). (c) Expression of inflammation related genes in peritoneal macrophages after the incubation with 100 μg/ml TRLs for 24 h and an 1 μg/ml LPS stimulation for the last 6 h (n = 4)

Fig. 3

TRLs increase ER stress and oxidative stress in HUVECs independent of ApoCIII. (a) Expression of ER stress related genes in HUVECs after the incubation of different doses of TRLs without ApoCIII (n = 4). (b) Proteins related to ER stress in HUVECs after the incubation of different doses of TRLs without ApoCIII (n = 4). (c) Expression of ER stress related genes in HUVECs after the incubation of TRLs without or without ApoCIII (n = 4). *p < 0.05 vs. PBS. (d) Western blot images of GRP78 after the incubation of TRLs with or without ApoCIII (n = 4)

Fig. 4

TRLs increase ER stress and oxidative stress in macrophages and VSMCs independent of ApoCIII. (a) Western blot images (up) and the protein quantification (down) of PDI and PERK in VSMCs after 48 h incubation with TRLs (100 μg/ml TG concentration). Expression of ER stress related genes (b) and oxidative stress related genes (c) in peritoneal macrophages after the incubation with TRLs (n = 4), *p < 0.05 vs. PBS. (d) Western blot images (up) and the protein quantification (down) of Nox4 in macrophages after the incubation of TRLs with or without ApoCIII (n = 4)