Aspirin-triggered lipoxin and resolvin E1 modulate vascular smooth muscle phenotype and correlate with peripheral atherosclerosis

Abstract

Atherosclerosis is a chronic inflammatory disease of the vessel wall. Recent evidence suggests that chronic vascular inflammation ensues as an imbalance between pro- and anti-inflammatory mediators. Recently identified lipid mediators (eg, lipoxins and resolvins) play active roles in promoting the resolution of inflammation. Alterations in vascular smooth muscle cell (VSMC) phenotype, which manifest as a loss of contractile protein expression and increased proliferation and migration, are prominent mechanistic features of both atherosclerosis and restenosis following various interventions (eg, angioplasty and bypass grafting). We sought to determine whether human atherosclerosis is associated with a "resolution deficit" and whether lipoxins and resolvins influence VSMC phenotype. Here we report that plasma levels of aspirin-triggered lipoxin are significantly lower in patients with symptomatic peripheral artery disease than in healthy volunteers. Both aspirin-triggered lipoxin and resolvin E1 block platelet-derived growth factor-stimulated migration of human saphenous vein SMCs and decrease phosphorylation of the platelet-derived growth factor receptor-β. Importantly, receptors for aspirin-triggered lipoxin and resolvin E1 (ALX and ChemR23, respectively) were identified in human VSMCs. Overall, these results demonstrate that stimulatory lipid mediators confer a protective phenotypic switch in VSMCs and elucidate new functions for these mediators in the regulation of SMC biology. These results also suggest that peripheral artery disease is associated with an inflammation-resolution deficit and highlight a potential therapeutic opportunity for the regulation of vascular injury responses.

Figures

Figure 1

Serum levels of aspirin-triggered lipoxin A4 (ATL) inversely correlate with PAD severity in humans. Serum was prepared from healthy volunteers (n = 38) and patients with PAD (claudication, n = 88; critical limb ischemia, n = 73), and ATL levels were analyzed by enzyme-linked immunosorbent assay; *P < 0.05.

Figure 2

Identification of ALX and ChemR23 in human saphenous vein smooth muscle cells. A: RT-PCR products of ALX and ChemR23 with GAPDH shown as a positive control. Analysis of ChemR23 (B) and ALX (C) expression in HSVSMCs by flow cytometry using phycoerythrin-conjugated anti-ChemR23 and anti-ALX antibodies, respectively. Results are representative of at least three separate cell donors.

Figure 3

Pretreatment with aspirin-triggered lipoxin A4 (ATL) and resolvin E1 (RvE1) attenuates PDGF-stimulated VSMC migration. A: Inhibition of PDGF-BB (10 ng/ml)-stimulated HSVSMC migration by ATL (1–500 nmol/L; gray bars); imatinib (4 μmol/L; hatched bar) is shown as a positive control. B: ALX receptor antagonist Boc-1 (100 μmol/L) counteracts the effects of ATL on VSMC migration. C: Inhibition of PDGF-BB-stimulated VSMC migration by RvE1 (0.1–500 nmol/L). Results are presented as the mean ± SEM and are representative of three to six independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way analysis of variance.

Figure 4

PDGFR-β phosphorylation status is modulated by ATL and RvE1. HSVSMCs (in 0.5% serum) were treated with ATL, RvE1, or RvD1 alone or 15 minutes before the addition of PDGF-BB (10 ng/ml) and the phosphorylation status of PDGFR-β was determined by immunoblot analysis using phospho-tyrosine-specific antibodies after immunoprecipitation of total PDGFR-β. Results are representative of three independent experiments.