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. 2022 Jan 8;11(2):204.
doi: 10.3390/cells11020204.

Native and Oxidized Low-Density Lipoproteins Increase the Expression of the LDL Receptor and the LOX-1 Receptor, Respectively, in Arterial Endothelial Cells

Rusan Catar  1 , Lei Chen  1 , Hongfan Zhao  1 , Dashan Wu  1 , Julian Kamhieh-Milz  2 , Christian Lücht  1 , Daniel Zickler  1 , Alexander W Krug  3 , Christian G Ziegler  3 , Henning Morawietz  4 , Janusz Witowski  1   5
Affiliations

Native and Oxidized Low-Density Lipoproteins Increase the Expression of the LDL Receptor and the LOX-1 Receptor, Respectively, in Arterial Endothelial Cells

Rusan Catar et al. Cells. .

Abstract

Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors-classical LDLR and alternative LOX-1-in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis.

Keywords: AP-1; LDL; LDL receptor; LOX-1; NF-κB; atherosclerosis; endothelium.

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Conflict of interest statement

None of the authors have any conflicts of interest related to this manuscript.

Figures

Figure 1
Figure 1
Characterization of human umbilical artery endothelial cells (HUAECs) in culture. (A) HUAEC morphology; (B–E) HUAECs were analyzed by flow cytometry after staining either with antibodies against endothelial cell-specific biomarkers (as indicated in color) or with isotype control IgG (in black). Representative histograms are shown.
Figure 2
Figure 2
LDL oxidation. Three LDL fractions were assessed: LDL freshly isolated from EDTA-plasma (EDTA-LDL),) LDL further purified by dialysis (nLDL), and nLDL treated with CuSO4 for 24 h (oxLDL). The level of oxidation was measured by (A) ELISA (n = 6), (B) absorbance at 234 nm (n = 4), and (C) gel electrophoretic mobility (n = 3), as described in Methods, with n referring to a number of separate LDL preparations. In (C), an exemplary gel is shown. t-test mean +/− SD with * p < 0.05.
Figure 3
Figure 3
Cell viability and LDL uptake. (A) The percentage of viable cells was measured with the WST-8 assay following 16 h of incubation with either nLDL or oxLDL at 100 µg/mL; n = 5. (B) Cell apoptosis was assessed by DNA-fragmentation following exposure of HUAECs to 100 μg/mL nLDL/oxLDL for 24 h. DNA was separated by electrophoresis on a 0.8% agarose gel at 90 V. (C,D) Uptake of DiI-labeled LDL by HUAECs was assessed by fluorescence microscopy using 450 nm and 490 nm filters after 3 h of incubation with (C) 100 μg/mL of either DiI-nLDL or DiI-oxLDL or with (D) 100 μg/mL of DiI-nLDL in the presence or absence of a 100-fold excess of unlabeled oxLDL. Magnification 40×, scale 100 µm.
Figure 4
Figure 4
Effect of nLDL and oxLDL on LDLR and LOX-1 expression. (A–D) Total RNA was harvested from HUAECs treated with either nLDL or oxLDL, and analyzed for LDLR and LOX-1 mRNA expression by reverse transcription-PCR. The results are presented in relation to the expression levels in untreated control cells. In A and B, HUAECs were incubated in the presence or absence of 100 µg/mL of nLDL or oxLDL for the times indicated (n = 6). In C and D, HUAECs were incubated for 12 h with either nLDL or oxLDL at doses indicated (n = 6). (E,F): Total protein was extracted from HUAECs incubated with 100 µg/mL of either nLDL or oxLDL for 24 h or (F) 7 h, and expression of target proteins was measured by Western blotting. Asterisks and hash signs represent a significant difference compared to control cells and nLDL-treated cells, respectively. t-test mean +/− SD with vs. 1 h * p < 0.05 and # vs. nLDL p < 0.005 ns = not significant.
Figure 5
Figure 5
Effect of nLDL and oxLDL on ERK1/2 and p38 phosphorylation. HUAECs were stimulated with 100 μg/mL of either nLDL or oxLDL for 20 min and immediately analyzed by Western blotting for the presence of phosphorylated ERK1/2 (A) and p38 (B). t-test mean +/− SD with * p < 0.05.
Figure 6
Figure 6
Activation of LDLR and LOX-1 gene promoters by nLDL and oxLDL. HUAECs were transiently transfected with full-length promoter constructs for LDLR (A,B) or LOX-1 (C–E) and then stimulated with 100 μg/mL of nLDL or oxLDL for 3 h and analyzed for luciferase activity. In (B,D), cells were pre-treated for 1 h with 100 nM of PD184352 (ERK1/2 inhibitor), 100 nM of PD-169316 (p38 inhibitor) or vehicle prior to stimulation. In (E), cells were treated with the same dose of oxLDL (100 μg/mL), but generated in response to different concentrations of CUSO4 (10 µM vs. 50 µM). t-test mean +/− SD with * p < 0.05 and ns = not significant.
Figure 7
Figure 7
Identifying the role of AP1 in nLDL-induced LDLR expression and oxLDL-induced LOX-1 expression. (A,B) Nuclear extracts were obtained from HUAECs incubated for 3 h with either nLDL (A) or oxLDL (B), at doses as indicated, and analyzed by EMSA using consensus oligonucleotides for AP-1 binding. (C,D) HUAECs were preincubated for 1 h with or without the AP-1 inhibitor SR-11302 (5 µM) and then stimulated with 100 µg/mL of nLDL or oxLDL for 12 h and analyzed for LDLR mRNA (C) and LOX-1 mRNA (D) expression. t-test mean +/− SD with * p < 0.05 and ns = not significant.
Figure 8
Figure 8
The role of NF-κB in the LOX-1 promoter activation by oxLDL. (A,B) Nuclear extracts were obtained from HUAECs incubated for 6 h with EDTA-LDL (control), nLDL or oxLDL (all at 100 µg/mL) and analyzed by EMSA using oligonucleotides for NF-κB binding within the LDLR promoter (A) or the LOX-1 promoter (B). In (C), HUAECs were preincubated with or without siRNA for the subunit p65 of NFκB (10 µM) for 24 h and then stimulated with 100 µg/mL of oxLDL for 12 h and analyzed for LOX-1 mRNA expression. t-test mean +/− SD with * p < 0.05.
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