Lipoxin A4 mediates aortic contraction via RHOA/RHO kinase, endothelial dysfunction and reactive oxygen species

Abstract

Background: Lipoxin A4 (LXA4) is a biologically active product generated from arachidonic acid by lipoxygenase action. The production of lipoxins is enhanced by aspirin through acetylation of cyclooxygenase-2, via a mechanism known as 'aspirin-triggered lipoxin'. LXA4 has both anti-inflammatory and proinflammatory actions, the latter being related with reocclusion and restenosis after coronary angioplasty in patients treated with aspirin. However, little is known of the actions of LXA4 on the vasculature. We hypothesized that LXA4 promotes contractile responses and contributes to endothelial dysfunction.

Methods: We used aorta from Wistar rats to assess vascular function. Reactive oxygen species (ROS) production and contractile and regulatory proteins were investigated.

Results: LXA4 induced concentration-dependent contractions via formyl peptide receptor-2 activation and both RhoA/Rho kinase inhibitor and ROS scavenger decreased this contraction. Also, endothelium removal, and COX-2 and NAD(P)H oxidase inhibitors attenuate the LXA4-induced contraction. LXA4 potentiated phenylephrine-induced contraction and inhibited acetylcholine-induced relaxation. In the presence of LXA4, ROS production was increased and protein expression of RhoA, phospho-myosin light chain, COX-2 and p67phox was higher.

Conclusion: LXA4 has a functional role in the vasculature and may contribute to further vascular damage in conditions where its production is exacerbated, such as in angioplasty-associated complications treated with aspirin.

Figure 1

Concentration-responses curves to lipoxin A₄ (LXA₄) and vehicle (VEH), (A) or phenylephrine in the presence or absence of LXA₄ (10 nmol/L) (B). Effects of cyclosporin H (C, CsH, FPR-1 antagonist) and WRW4 (D, FPR-2 antagonist) on LXA₄-induced contraction. The results are expressed as the means ± SEM for the number of animals used (4-5). Two-way ANOVA: * represents statistically significant (p<0.05) vs. LXA₄.

Figure 2

Effects of Y27631 (RhoA/Rho kinase inhibitor) on LXA₄-induced contraction (A). Representative blots and densitometric analyses of protein expression of phosphorylated myosin light chain (pMLC) (B), RhoA (C) and β-actin of aorta from Wistar rats treated with lipoxin A₄ (LXA₄) (10 nmol/L) or vehicle (VEH) for 15 min. The results are expressed as the means ± SEM for the number of animals used (4-5). Twoway ANOVA or t-test: * represents statistically significant (p<0.05) vs. LXA₄.

Figure 3

Effects of endothelium removal (E-) on LXA₄-induced contraction (A). Concentration response curves to acetylcholine in the presence or absence of lipoxin A₄ (LXA₄, 10 nmol/L) (B). The results are expressed as the means ± SEM for the number of animals used (4-5). Two-way ANOVA * represents statistically significant (p<0.05) vs. LXA₄.

Figure 4

Effects of indomethacin (Indo, COX inhibitor) (A), NS-398 (COX-2 inhibitor) (B), SC-560 (COX-1 inhibitor) (C) or SQ-29548 (thromboxane A₂ and/or prostaglandin H₂ receptor antagonist) on LXA₄-induced contraction. Representative blots and densitometric analyses of protein expression of COX-1 (E), COX-2 (F) and β-actin of aorta from Wistar rats treated with lipoxin A₄ (LXA₄) (10 nmol/L) or vehicle (VEH) for 15 min. The results are expressed as the means ± SEM for the number of animals used (4-5). Two-way ANOVA or t-test: * represents statistically significant (p<0.05) vs. LXA₄.

Figure 5

Concentration-responses curves to lipoxin A₄ (LXA₄) and vehicle (VEH) (A) or arachidonic acid (AA) (B) in pre-contracted rings with phenylephrine in the presence or absence of indomethacin (INDO, 10 μmol/L) (B). Effects of INDO (C) on concentration-responses curves to AA in non-pre-contrated rings.

Figure 6

Effects of tempol (superoxide anion scavenger) (A) or apocynin (NAD(P)H oxidase inhibitor) (C) on LXA₄-induced contraction. Effect of LXA₄ (10 μmol/L) on reactive oxygen species (ROS) generation (B). Upper panel: representative fluorescence photomicrographs of microscopic sections from mesenteric resistance arteries. Lower panel: densitometric analysis of fluorescence intensity. Representative blots and densitometric analyses from protein expression for p67phox (D) and β-actin of aorta from Wistar rats treated with lipoxin A₄ (LXA₄) (10 nmol/L) or vehicle (VEH) for 15 min. The results are expressed as the means ± SEM for the number of animals used (4-5). Two-way ANOVA or t-test: * represents statistically significant (p<0.05) vs. LXA₄.

Figure 7

Proposed effects of lipoxin A₄ (LXA₄) on vascular contraction. Formyl peptide receptor-2 (FPR-2); Cyclooxygenase-2 (COX-2); reactive oxygen reactive (ROS); nitric oxide (NO); NAD(P)H oxidase, Rho-associated protein kinase (ROCK), Ras homolog gene family, member A (RhoA), phosphorylated myosin light chain (pMLC).