Lipocalin (LCN) 2 Mediates Pro-Atherosclerotic Processes and Is Elevated in Patients with Coronary Artery Disease

Abstract

Background: Lipocalin (LCN) 2 is associated with multiple acute and chronic inflammatory diseases but the underlying molecular and cellular mechanisms remain unclear. Here, we investigated whether LCN2 is released from macrophages and contributes to pro-atherosclerotic processes and whether LCN2 plasma levels are associated with the severity of coronary artery disease progression in humans.

Methods and results: In an autocrine-paracrine loop, tumor necrosis factor (TNF)-α promoted the release of LCN2 from murine bone-marrow derived macrophages (BMDM) and vice versa. Moreover, LCN2 stimulation of BMDM led to up-regulation of M1 macrophage markers. In addition, enhanced migration of monocytic J774A.1 cells towards LCN2 was observed. Furthermore, LCN2 increased the expression of the scavenger receptors Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) as well as scavenger receptor class A-1 (SRA-1) and induced the conversion of macrophages to foam cells. In atherosclerotic lesions of low density lipoprotein receptor-deficient (ldlr-/-) mice fed a high fat, high cholesterol diet, LCN2 was found to be co-localized with macrophages in the shoulder region of the atherosclerotic plaque. In addition, LCN2 plasma levels were significantly increased in plasma samples of these mice. Finally, LCN2 plasma levels correlated with the severity of coronary artery disease (CAD) in patients as determined by coronary angiography.

Conclusions: Here we demonstrated that LCN2 plays a pivotal role in processes involved in atherogenesis by promoting polarization and migration of monocytic cells and development of macrophages towards foam cells. Moreover, LCN2 may be used as a prognostic marker to determine the status of CAD progression.

Fig 1. TNF-α enhances LCN2 expression and secretion in macrophages.

(A) LCN2 release from murine primary BMDM following oxLDL (50 μg/mL), IL-6 (200 ng/mL) or TNF-α (50 ng/mL) stimulation was determined by ELISA at the indicated time points. LPS (100 ng/mL for 6 hours) was used as positive control. *P<0.05, **P<0.01 vs. control, n = 6–8 replicated experiments. (B) LCN2 mRNA expression following TNF-α (50 ng/mL) stimulation was determined by real time PCR at the indicated time points. LPS (100 ng/mL for 6 hours) was used as positive control. HPRT was used as housekeeping gene and the relative mRNA expression was calculated using the 2^-ΔΔCT method. *P<0.05, **P<0.01 vs. control, n = 4–7 replicated experiments. (C) LCN2 protein expression following TNF-α (50 ng/mL) stimulation for 6 hours was determined by Western blot. LPS was used as positive control. GAPDH was used as loading control. **P<0.01 vs. control, n = 5 replicated experiments.

Fig 2. LCN2 induces cell migration of monocytic cells.

Migration of murine monocyte/macrophage-like J774A.1 cells in response to LCN2 (0.5 μg/mL) was carried out in transwell cell culture inserts for 24 hours. Medium with 10% FCS was used as positive control. Migration is depicted as induction over unstimulated control. *P<0.05, **P<0.01 vs. control, n = 5 replicated experiments.

Fig 3. LCN2 enhances mRNA expression of M1 macrophage markers.

(A) mRNA expression of the M1 macrophage markers TNF-α, iNOS, IL-6 and CCL5 as well as the M2 macrophage markers Ym1/2 and Arg1 in murine primary BMDM following LCN2 (0.5 μg/mL) stimulation was determined by real time PCR at the indicated time points. HPRT was used as housekeeping gene and the relative mRNA expression was calculated using the 2^-ΔΔCT method. con = control. *P<0.05, **P<0.01 vs. control, n = 5–10 replicated experiments. (B) Release of TNF-α from murine primary BMDM following LCN2 (0.5 μg/mL) stimulation for 6 hours was assessed by ELISA. *P<0.05 vs. control, n = 12. (C) Protein expression of the M2 macrophage markers Ym1 and Arg1 following LCN2 (0.5 μg/mL) stimulation for 6 hours was determined by Western blot. GAPDH was used as loading control. n = 3–4 replicated experiments.

Fig 4. LCN2 induces foam cell formation.

Foam cell formation is illustrated by the uptake of Dil-labelled oxLDL (10 μg/mL) for 4 hours. Murine primary BMDM were pre-stimulated with LCN2 (0.5 μg/mL) for 24 hours. Foam cell formation is given as percentage of Dil-positive cells per high power field. Representative pictures are shown. Scale bars = 200 μm. Inserts show a higher magnification of the pictures. Scale bars in insert = 50 μm. *P<0.05 vs. control, n = 6 replicated experiments.

Fig 5. LCN2 modulates the mRNA expression of scavenger receptors in macrophages.

The mRNA expression of the oxLDL receptors LOX-1, SRA-1, SRB-1 and CD36 in murine primary BMDM following LCN2 (0.5 μg/mL) stimulation was determined by real time PCR at the indicated time points. HPRT was used as housekeeping gene and the relative mRNA expression was calculated using the 2^-ΔΔCT method. *P<0.05, **P<0.01 vs. control, n = 6–10 replicated experiments.

Fig 6. Hypercholesterolemic mice reveal pronounced LCN2 levels in blood plasma and atherosclerotic lesions.

(A) LCN2 plasma levels of ldlr ^−/− mice fed a high fat, high cholesterol diet (HCD) and of ldlr ^−/− mice fed a normal chow diet (chow) over a period of 24 weeks were quantified by ELISA. **P<0.01 vs. control, n = 5–10 animals. (B) Consecutive cross sections of the aortic root dissected from ldlr ^−/− mice 24 weeks after feeding a high fat, high cholesterol diet were stained for LCN2 and Picrosirius Red. For the distinction of different collagen fibres pictures were taken using polarized light. Scale bar = 100 μm. (C) Cross sections of the aortic root from ldlr ^−/− mice 24 weeks after feeding a high fat, high cholesterol diet were stained for LCN2 or MOMA-2 or double stained for LCN2 and MOMA-2. Sections were counterstained with hematoxylin. Scale bars = 100 μm. Representative pictures are shown.

Fig 7. Patients with coronary artery disease have higher LCN2 plasma levels.

Plasma samples from 98 patients undergoing coronary angiography were analyzed for LCN2 by ELISA. (A) Patients were classified as controls without CAD (n = 20) and with CAD (n = 78). (B) Subgroup analysis of control patients without CAD (n = 20) and patients subdivided in 1-vessel (CAD 1, n = 24), 2-vessel (CAD 2, n = 27) and 3-vessel (CAD 3, n = 27) CAD. *P<0.05, **P<0.01 vs. control, ^#P<0.05 vs. CAD2. Box plots of median with 25^th/75^th percentiles and whiskers with 10^th/90^th percentiles are shown.