Aspirin triggered-lipoxin A4 reduces the adhesion of human polymorphonuclear neutrophils to endothelial cells initiated by preeclamptic plasma

Abstract

Introduction: Preeclampsia is a disorder of pregnancy, characterized by hypertension and proteinuria after 20 weeks of gestation. Here, we evaluated the role of aspirin triggered-lipoxin A(4) (ATL, 15-epi-LXA(4)) on the modulation of the adhesion of human polymorphonuclear neutrophils (PMN) to endothelial cells initiated by preeclamptic plasma.

Materials and methods: Plasma from preeclamptic, normotensive pregnant, and non-pregnant women were analyzed for factors involved in regulating angiogenesis, inflammation and lipid peroxidation. Plasma from preeclamptic women was added to human umbilical vein endothelial cells, and the adhesion of PMN (incubated with or without ATL) to cells was evaluated.

Results: Preeclampsia was associated with some augmented anti-angiogenic, oxidative and pro-inflammatory markers, as well as increasing human PMN-endothelial cell adhesion. This cell adhesion was reduced when human PMN were incubated with ATL prior to addition to endothelial monolayers.

Discussions and conclusions: Our results are the starting point for further research on the efficacy and rational use of aspirin in preeclampsia.

Figure 1. Plasma levels of pro-angiogenic and pro-inflammatory mediators are elevated in preeclamptic women

Plasma was obtained from non-pregnant (n=11), normotensive pregnant (n=15) and preeclamptic pregnant (n=14) women and inflammatory markers and angiogenic factors were analyzed by Luminex. Data were normalized by quantification of plasma protein. *P<0.05, **P<0.01,***P<0.001.

Figure 2. Lipid mediator levels are modulated in women with preeclampsia

Plasma was obtained from non-pregnant (n=13), normotensive pregnant (n=17) and preeclampitc pregnant (n=14) women and analyzed by enzyme–linked immunosorbent assay. *P<0.05, **P<0.01,***P<0.001.

Figure 3. Systemic oxidative stress is elevated in preeclamptic patients

Lipid peroxidation levels as measured by TBARS (A), and 8-isoprostane (B) were measured in plasma of women with preeclampsia (n=12) as compared with normotensive pregnant women (n=15) and non-pregnant women (n=8). *P<0.05, **P<0.01,***P<0.001.

Figure 4. Aspirin-triggered lipoxin (ATL) reduces leukocyte-endothelial interactions initiated by preeclamptic plasma, 8-isoprostane and TBARS: Endothelial and leukocyte directed actions

HUVEC cells were stimulated overnight with IL-1β (positive control, 10 ng/mL), plasma from normotensive pregnant women (n=4) or preeclamptic women (n=8). Then, PMN labeled with CFDA-SE were added to pre-stimulated HUVEC cells, and cell adhesion was assessed after 15min as described in methods (A). On the other hand, HUVEC were stimulated overnight with 8-isoprostane (0.1–1000 nM) (C) and TBARS (1–100.000 nM) (E). Human PMN were incubated with ATL (10nM; 15min) prior to addition to stimulated HUVEC monolayers with plasma, 8-isoprostane and TBARS, respectively (B,D,F). Results are mean ± s.e.m *P<0.05, **P<0.01,***P<0.001

Figure 5. Isoprostane and TBARS stimulate PMN adhesion to HUVEC

Human PMN were stimulated with Leukotriene B₄ (positive control, 10 nM; 30min), 8-isoprostane (1–1000 nM; 30min) or TBARS (1–1000 nM; 30min) and adhesion to non-stimulated HUVEC was assessed after 15min (A). Human PMN were incubated with ATL (10nM; 15min) prior to addition to stimulated HUVEC monolayers (B). *P<0.05, **P<0.01,***P<0.001

Figure 6. Proposed function of 15-epi-Lipoxin A4 (ATL) on maternal vascular endothelium response to placenta derived substances

In pregnancy placenta generated pro-inflammatory cytokines (PIC), reactive oxidative stress molecules (ROS) and syncytiotrophoblast microfragments gain access to the maternal systemic circulation. These molecules stimulate inflammation of the maternal vascular endothelium, the production of ROS, PIC, and vasoactive prostaglandins (PG) leading to endothelial activation, polymorphonuclear neutrophil (PMN) adhesion and transmigration and vascular leakage. This sets the stage for a feed forward cyclic process in which mediators from the endothelium stimulate the placenta to produce ROS, PIC and other inflammatory mediators. In preeclampsia this theoretical scenario is increased as compared to normal pregnancies leading to adverse clinical manifestations. ATL may be beneficial in the treatment of preeclampsia by decreasing the oxidative and pro-inflammatory responses and their downstream consequences.