The 5A apolipoprotein A-I mimetic peptide displays antiinflammatory and antioxidant properties in vivo and in vitro

Abstract

Objective: The apolipoprotein (apo)A-I mimetic peptide 5A is highly specific for ATP-binding cassette transporter (ABC)A1-mediated cholesterol efflux. We investigated whether the 5A peptide shares other beneficial features of apoA-I, such as protection against inflammation and oxidation. Methods- New Zealand white rabbits received an infusion of apoA-I, reconstituted high-density lipoprotein (HDL) containing apoA-I ([A-I]rHDL), or the 5A peptide complexed with phospholipids (1-palmitoyl-2-linoleoyl phosphatidylcholine [PLPC]), before inserting a collar around the carotid artery. Human coronary artery endothelial cells (HCAECs) were incubated with (A-I)rHDL or 5A/PLPC before stimulation with tumor necrosis factor alpha. Results- ApoA-I, (A-I)rHDL, and 5A/PLPC reduced the collar-mediated increase in (1) endothelial expression of cell adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1; (2) production, as well as the expression of the Nox4 catalytic subunits of the NADPH oxidase; and (3) infiltration of circulating neutrophils into the carotid intima-media. In HCAECs, both 5A/PLPC and (A-I)rHDL inhibited tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, as well as the nuclear factor kappaB signaling cascade and production. The effects of the 5A/PLPC complex were no longer apparent in HCAECs knocked down for ABCA1.

Conclusions: Like apoA-I, the 5A peptide inhibits acute inflammation and oxidative stress in rabbit carotids and HCAECs. In vitro, the 5A peptide exerts these beneficial effects through interaction with ABCA1.

Figure 1

Immunohistochemical staining for VCAM-1, ICAM-1 and CD18 in representative non-collared and collared carotid artery sections from NZW rabbits infused with either saline, the 5A/PLPC complex (20mg/kg), lipid-free apoA-I (8mg/kg), or (A-I)rHDL 24 hours prior to collar implantation. Histograms: endothelial expression of VCAM-1, ICAM-1 and infiltration of neutrophils (CD18) into the intima-media were quantified as described in Methods. Results are expressed as mean ±SEM (*p<0.05 vs. saline, ** p<0.01 vs. no collar, n=6 per group).

Figure 2

(A) mRNA levels of Nox 2 and Nox 4 in non-collared and collared carotid artery sections from NZW rabbits infused with either saline, the 5A/PLPC complex (20mg/kg of 5A) or lipid-free apoA-I (8mg/kg), 24 hours prior to collar implantation. (B) ROS generation was detected using dihydroethidum (DHE) fluorescence in non-collared and collared carotid artery sections from NZW rabbits infused with either saline, the 5A/PLPC complex (20mg/kg of 5A) or lipid-free apoA-I (8mg/kg), as described in Methods. (*p<0.05 vs. non-collar, **p<0.05 vs. saline, n=6 per group). Results are expressed as mean expression levels of Nox 2, or Nox 4, normalized to 18S, ±SEM (*p<0.01 vs. Non-Collar, **p<0.05 vs. saline, n=6 per group).

Figure 3

(A-B) Flow cytometry analysis of TNFα-induced VCAM-1 (panel A) and ICAM-1 (panel B) expression in HCAECs exposed to either PBS, or increasing concentrations of (A-I)rHDL, or increasing concentrations of the 5A/PLPC complex for 16 hours. (C-D) Flow cytometry analysis of TNFα-induced VCAM-1 (panel C) and ICAM-1 (panel D) expression in HCAECs exposed to either PBS, or 1mg/mL (A-I)rHDL, or 1mg/mL 5A/PLPC for 2, 12, 16 or 24 hours. Changes in mean fluorescence of cell surface VCAM-1 and ICAM-1 expression are expressed as mean±SEM of 4 independent experiments (#p<0.01 vs. control (C), *p<0.05 vs. PBS).

Figure 4

(panels A & B) Immunoblot analysis of TNFα-induced ICAM-1 and VCAM-1 expression in HCAECs exposed to PBS, (A-I)rHDL (final apoA-I concentration 1mg/mL), or the 5A/PLPC complex (final peptide concentration 1mg/mL) for 16 hours. Relative quantification of ICAM-1 and VCAM-1 normalized to β-actin is expressed as mean±SEM of 3 independent experiments performed in triplicate (*p<0.05 vs. control (C), **p<0.05 vs. PBS). (panels C&D) Immunoblot analysis of the nuclear translocation of NF-κB p65 subunit and of the phosphorylation of IκB in HCAEC cells exposed to PBS, 1mg/mL (A-I)rHDL, or 1mg/mL 5A/PLPC for 16 hours and stimulated with TNFα for 10 min. Relative quantification of NF-κB and P-IκB normalized to total IκB is expressed as mean±SEM of 3 independent experiments performed in triplicate (*p<0.05 vs. control (C), **p<0.05 vs. PBS). Experiments shown in panels B &D were performed in HCAECs knocked down for ABCA1.

Figure 5

TNFα induced ROS production in HCAECs exposed to PBS, (A-I)rHDL (Final apoA-I concentration 1mg/mL), or the 5A/PLPC complex (final peptide concentration 1mg/mL) for 16 hours. ROS generation was detected using dihydroethidum (DHE) fluorescence. (A) Nuclear DHE fluorescence in HCAECs. (B) Nuclear DHE fluorescence in HCAECs transfected with ABCA1 siRNA (50nM). Results are expressed as mean±SEM of three independent experiments performed in triplicates (*p<0.05 vs. control (C), **p<0.05 vs. PBS).