Inhibition of Vascular Inflammation by Apolipoprotein A-IV

Abstract

Background: Apolipoprotein (apo) A-IV, the third most abundant apolipoprotein in human high density lipoproteins (HDLs), inhibits intestinal and systemic inflammation. This study asks if apoA-IV also inhibits acute vascular inflammation.

Methods: Inflammation was induced in New Zealand White rabbits by placing a non-occlusive silastic collar around the common carotid artery. A single 1 mg/kg intravenous infusion of lipid-free apoA-IV or saline (control) was administered to the animals 24 h before collar insertion. The animals were euthanised 24 h post-collar insertion. Human coronary artery cells (HCAECs) were pre-incubated with reconstituted HDLs containing apoA-IV complexed with phosphatidylcholine, (A-IV)rHDLs, then activated by incubation with tumour necrosis factor (TNF)-α. Cell surface vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the TNF-α-activated HCAECs was quantified by flow cytometry. VCAM-1, ICAM-1 and 3β-hydroxysteroid-Δ24 reductase (DHCR24) mRNA levels were quantified by real time PCR.

Results: Apolipoprotein ApoA-IV treatment significantly decreased collar-induced endothelial expression of VCAM-1, ICAM-1 and neutrophil infiltration into the arterial intima by 67.6 ± 9.9% (p < 0.01), 75.4 ± 6.9% (p < 0.01) and 74.4 ± 8.5% (p < 0.05), respectively. It also increased endothelial expression of DHCR24 by 2.6-fold (p < 0.05). Pre-incubation of HCAECs with (A-IV)rHDLs prior to stimulation with TNF-α inhibited VCAM-1 and ICAM-1 protein levels by 62.2 ± 12.1% and 33.7 ± 5.7%, respectively. VCAM-1 and ICAM-1 mRNA levels were decreased by 55.8 ± 7.2% and 49.6 ± 7.9%, respectively, while DHCR24 mRNA expression increased by threefold. Transfection of HCAECs with DHCR24 siRNA attenuated the anti-inflammatory effects of (A-IV)rHDLs. Pre-incubation of TNF-α-activated HCAECs with (A-IV)rHDLs also inhibited nuclear translocation of the p65 subunit of nuclear factor-κB (NF-κB), and decreased IκBα phosphorylation.

Conclusion: These results indicate that apoA-IV inhibits vascular inflammation in vitro and in vivo by inhibiting NF-κB activation in a DHCR24-dependent manner.

Keywords: 3β-hydroxysteroid-Δ24 reductase; apolipoprotein A-IV; endothelial cells; high-density lipoproteins; inflammation; nuclear factor-kappaB.

FIGURE 1

Apolipoprotein A-IV inhibits acute vascular inflammation in NZW rabbit carotid arteries. NZW rabbits (n = 6/group) were infused iv with saline [Panels (A,B,E,F,I,J,M,N)], apoA-I [Panels (C,G,K,O); 8 mg/kg iv] or apoA-IV [Panels (D,H,L,P); 1 mg/kg iv] 24 h prior to inserting a non-occlusive peri-arterial collar around the left common carotid artery. The animals were euthanised 24 h post-collar insertion. Sections from the non-collared [Panels (A,E,I,M)] and the collared carotid arteries were stained for neutrophils [CD18, Panels (B–D)], ICAM-1 [Panels (F–H)], VCAM-1 [Panels (J–L)] and DHCR24 [Panels (N–P)]. Representative immunostained images as shown. *p < 0.05, **p < 0.01.

FIGURE 2

A single apoA-IV infusion does not alter the anti-inflammatory properties of rabbit HDLs. HDLs were ultracentrifugally isolated from NZW rabbits that had been treated with saline [Panels (A,D)], lipid-free apoA-I [Panels (B,E)] or lipid-free apoA-IV [Panels (C,F)] as described in the legend to Figure 1. The isolated HDLs (final apoA-I concentration: 5, 10, or 20 μM) were incubated at 37°C for 16 h with HCAECs prior to stimulation with TNF-α (0.2 ng) for 5 h at 37°C. ICAM-1 and VCAM-1 protein expression was quantified by flow cytometry. Data points represent mean ± SEM of three independent experiments with three replicates/experiment. *p < 0.05, ***p < 0.001.

FIGURE 3

Size and composition of discoidal (A-IV)rHDLs and discoidal (A-I)rHDLs. Discoidal (A-IV)rHDLs and (A-I)rHDLs were prepared by the cholate dialysis method and analysed in terms of size and composition [Panel (A)]. The discoidal rHDLs were then cross-linked with BS and their migration was compared with that of cross-linked lipid-free apoA-I and apoA-IV [Panel (B)].

FIGURE 4

(A-IV)rHDLs inhibit ICAM-1, VCAM-1 expression and increase DHCR24 expression in TNF-α-stimulated HCAECs. HCAECs were incubated for 16 h with (A-I)rHDLs (final apoA-I concentration 32 μM) and (A-IV)rHDLs (final apoA-IV concentrations 5 and 10 μM). The rHDLs were then removed and the cells were incubated for a further 5 h with TNF-α (final concentration 0.2 ng/mL). ICAM-1 [Panel (A)] and VCAM-1 [Panel (B)] protein expression was quantified by flow cytometry. ICAM-1 [Panel (C)], VCAM-1 [Panel (D)], and DHCR24 [Panel (E)] mRNA levels were quantified by qPCR. The mean ± SEM of three independent experiments, each performed in triplicate are shown. *p < 0.05, ***p < 0.005.

FIGURE 5

(A-IV)rHDL inhibits NF-κB activation in TNF-α-stimulated HCAECs. HCAECs were incubated for 16 h at 37°C with (A-I)rHDLs (final apoA-I concentration 32 μM) or (A-IV)rHDLs (final A-IV concentration 5 and 10 μM). The rHDLs were removed and the cells were incubated for 20 min at 37°C with TNF-α (0.2 ng/mL). Nuclear extracts were isolated and subjected to immunoblotting using β-actin as a loading control. Nuclear p65 protein levels (A,B) and cytoplasmic levels of p65 and phosphorylated IκBα (pIκBα), (C,D) are shown. (E) Shows the ratio of cytoplasmic pIκBα/IκBα. Results represent mean ± SEM of three independent experiments, each performed in triplicate. **p < 0.01, ***p < 0.001.

FIGURE 6

(A-IV)rHDLs inhibit TNF-α-induced ICAM-1 and VCAM-1 expression in HCAECs in a DHCR24-dependent manner. HCAECs were transfected with DHCR24 or scrambled siRNA then pre-incubated at 37°C for 16 h with (A-I)rHDLs (final apoA-I concentration 32 μM) or (A-IV)rHDLs (final apoA-IV concentration 10 μM). The rHDLs were removed and the cells were stimulated for 5 h with TNF-α (final concentration 0.2 ng/mL). The reduction in DHCR24 protein and mRNA levels in the transfected HCAECs was quantified by Western blotting and qPCR [Panel (A)]. ICAM-1 [Panel (B)] and VCAM-1 [Panel (C)] protein levels were quantified in the TNF-α-activated HCAECs by flow cytometry. Results represent the mean ± SEM of three independent experiments, each performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs. TNF-α-stimulated cells.

FIGURE 7

Lipid-free and lipid-associated apoA-IV reduces acute vascular inflammation by inhibiting endothelial ICAM-1 and VCAM-1 expression (A,B) and NF-κB activation in a DHCR24-dependent manner (C).