A novel nitroalkene-α-tocopherol analogue inhibits inflammation and ameliorates atherosclerosis in Apo E knockout mice

Abstract

Background and purpose: Atherosclerosis is characterized by chronic low-grade inflammation with concomitant lipid accumulation in the arterial wall. Anti-inflammatory and anti-atherogenic properties have been described for a novel class of endogenous nitroalkenes (nitrated-unsaturated fatty acids), formed during inflammation and digestion/absorption processes. The lipid-associated antioxidant α-tocopherol is transported systemically by LDL particles including to the atheroma lesions. To capitalize on the overlapping and complementary salutary properties of endogenous nitroalkenes and α-tocopherol, we designed and synthesized a novel nitroalkene-α-tocopherol analogue (NATOH) to address chronic inflammation and atherosclerosis, particularly at the lesion sites.

Experimental approach: We synthesized NATOH, determined its electrophilicity and antioxidant capacity and studied its effects over pro-inflammatory and cytoprotective pathways in macrophages in vitro. Moreover, we demonstrated its incorporation into lipoproteins and tissue both in vitro and in vivo, and determined its effect on atherosclerosis and inflammatory responses in vivo using the Apo E knockout mice model.

Key results: NATOH exhibited similar antioxidant capacity to α-tocopherol and, due to the presence of the nitroalkenyl group, like endogenous nitroalkenes, it exerted electrophilic reactivity. NATOH was incorporated in vivo into the VLDL/LDL lipoproteins particles to reach the atheroma lesions. Furthermore, oral administration of NATOH down-regulated NF-κB-dependent expression of pro-inflammatory markers (including IL-1β and adhesion molecules) and ameliorated atherosclerosis in Apo E knockout mice.

Conclusions and implications: In toto, the data demonstrate a novel pharmacological strategy for the prevention of atherosclerosis based on a creative, natural and safe drug delivery system of a non-conventional anti-inflammatory compound (NATOH) with significant potential for clinical application.

Scheme 1

Synthesis of NATOH. Reagents and conditions: (a) (i) Br₂, hexane anh. rt; (ii) Ac₂O, AcOH, H₂SO₄, rt, 72%; (b) N‐methylmorpholine N‐oxide, CH₃CN, rt, 93%; and (c) CH₃NO₂, AcONH₄, reflux, 27%.

Figure 1

Electrophilic properties of NATOH. (A) NATOH (100 μM) was dissolved in phosphate buffer (20 mM) pH 7.4, 1% SDS and incubated with BME (1 mM). Spectra of the reaction were obtained in the 220–660 nm range every 60 s. Scans shown were taken every 3 min. Inset: Exponential decay of the reaction followed at 350 nm. (B) RP‐HPLC characterization of the reaction between NATOH (10 nmol) and BME (375 nmol) at pH 7.4 or 2.0 (λ = 295 nm). (C) RP‐HPLC characterization of the reaction between α‐TOH (10 nmol) and BME (375 nmol) at pH 7.4 or 2.0 (λ = 295 nm).

Figure 2

Electrophilic properties of NATOH in human LDL or in liposomes. (A) NATOH (100 μM) was incorporated into human LDL (see protocol below) or (B) into POPG : POPC liposomes (200 μM), and the reaction with BME was followed by UV–Vis spectrophotometry. Each spectrum was taken every 1 min for 15 min.

Figure 3

α‐TOH and NATOH (50 μM) in liposomes (POPG + POPC, 200 μM) were incubated with FL (1 μM) in phosphate buffer (20 mM) pH 7.4, 1% SDS for 30 min at 37°C. Then, the mixture was incubated with the free radical generator AAPH (12 mg·mL⁻¹) and the antioxidant capacity were measured by the ORAC technique with a fluorometric assay using λex = 485 nm and λem = 518 nm in a Thermo Scientific™ Varioskan LUX multimode microplate reader. The values shown are means ± SD.

Figure 4

(A) Effects of NATOH on the subcellular localization of NF‐κB/p65 subunit in THP‐1 macrophages analysed by immunofluorescence microscopy. Cells were treated with/without NATOH/α‐TOH (50 μM) in liposomes overnight and then activated with LPS (1 μg·mL⁻¹). In the negative control (control), cells were not treated with LPS. Scale bar = 10 μm. (B) Upper panel: Binary quantification of nuclear fluorescence intensity (NFI, nuclear translocation of p65) versus total fluorescence intensity (TFI). (B) Lower panel: NATOH inhibits LPS‐induced IL‐1β secretion by THP‐1. Differentiated macrophages were pretreated overnight with NATOH or α‐TOH as before. Then, cells were stimulated with LPS (100 ng·mL⁻¹, 3 h) and after with ATP (5 mM, 45 min). IL‐1β was then measured by elisa in the supernatant. The values are showed as mean ± SEM. (C and D, upper panels) NF‐κB‐dependent gene expression and protein translation in macrophages. THP‐1 macrophages were treated with/without NATOH/α‐TOH in liposomes as before. Cells were then stimulated with LPS (100 ng·mL⁻¹, 4 h), and IL‐6 and MCP‐1 were analysed by qPCR. (C and D, bottom panels) Raw 264.7 murine macrophages were treated with/without NATOH/α‐TOH in liposomes for 8 h. Media were then removed, and cells were stimulated overnight with LPS (50 ng·mL⁻¹). Proteins of interest were measured by elisa in the supernatant.

Figure 5

NATOH induces Nrf2‐Keap1 cytoprotective response in human macrophages. THP‐1 cells were differentiated into macrophages and then treated overnight with different doses of NATOH or α‐TOH in liposomes or unloaded liposomes. mRNA was extracted, and the relative expression of HO‐1 and GCLM was measured by qPCR. Results are expressed as the mean ± SEM of three independent experiments.

Figure 6

Incorporation of NATOH into human lipoproteins. Human plasma was treated with NATOH (25, 50 or 100 μM; B–D) for 6 h at 37°C and the LDL lipoprotein fraction isolated and extracted with methanol (9:1, v:v). Control was performed with non‐treated plasma (A). Samples were analysed by RP‐HPLC using δ‐TOH as an internal standard. In all panels, the black line indicates absorbance at 295 nm and red line absorbance at 350 nm.

Figure 7

Detection of NATOH in the plasma of Apo E knockout mice. (A–C) Mice fed with an HFD without extra addition of vitamin E were administered, by gavage, daily vehicle (A), α‐TOH (B) or NATOH (C), 200 mg·kg⁻¹·day⁻¹ for 14 days. Mice were killed, and plasma samples taken 12 h post‐last administration were extracted with methanol and analysed by RP‐HPLC (A–C; red line Abs. at 350 nm; black line Abs. at 295 nm). (D) A calibration curve of each of the analytes was generated to quantify the concentration of α‐TOH and NATOH in the plasma of mice. (E) Table showing the concentrations of α‐TOH and NATOH in both control and treated mice. ND, not detected.

Figure 8

Detection of NATOH in albumin/HDL (A, C and E) or in VLDL/LDL lipoprotein fractions (B, D and F) of Apo E knockout mice. The albumin/HDL fraction (bottom fraction) and VLDL/LDL lipoprotein fractions were isolated by ultracentrifugation (as described in the Methods section), and the presence of NATOH or α‐TOH was analysed as before. (F) Inset shows the same sample that was spiked with 0.05 nmol of NATOH (blue line).

Figure 9

Detection of NATOH by RP‐HPLC in aortas from HFD‐fed Apo E knockout mice. (A) Standard elution profile of NATOH by RP‐HPLC detected at 350 nm. (B) Elution profile of the aortic arch of vehicle‐treated mice (100 μL sunflower oil). (C) Elution profile of the aortic arch of NATOH‐treated mice (200 mg·kg⁻¹·day⁻¹ in vehicle). (D) Elution profile of the abdominal aorta of vehicle‐treated mice. (E) Elution profile of the abdominal aorta of NATOH‐treated mice.

Figure 10

NATOH reduces atherosclerosis in HFD‐treated Apo E knockout mice without affecting weight gain. (A) Apo E knockout mice were treated with NATOH or α‐TOH via gavage (100 mg·kg⁻¹ in 100 μL sunflower oil) from Monday to Friday for 14 weeks. Mice were killed, aorta dissected and atherosclerotic plaque formation was manually measured using the Oil Red O staining and ImageJ software. (B) Atherosclerotic plaque area, number and lesion size. Plaque area was determined as a percentage of total aortic area, *P < 0.05 (t‐test), Nr of lesions P < 0.05. (C) Weight gain versus time and plasma cholesterol level in NATOH and tocopherol‐treated animals. (D) IL‐1β, ICAM‐1, VCAM‐1 and TNF‐α mRNA were down‐regulated by NATOH in vivo. The aorta of Apo E knockout mice treated as described in (A) was extracted, and the RNA was isolated using TRIzol. Levels of IL‐1β, VCAM‐1, ICAM‐1 and TNF‐α were measured by RT‐qPCR using GAPDH as a housekeeping gene. One‐tailed t‐test: *P < 0.05.