Circulating Neutrophils Do Not Predict Subclinical Coronary Artery Disease in Women with Former Preeclampsia

Abstract

: Introduction: Preeclampsia (PE) represents a hypertensive pregnancy disorder that is associated with increased cardiovascular disease (CVD) risk. This increased risk has been attributed to accelerated atherosclerosis, with inflammation being a major contributor. Neutrophils play an important role in the onset and progression of atherosclerosis and have been associated with vascular damage in the placenta as well as the chronic inflammatory state in women with PE. We therefore investigated whether circulating neutrophil numbers or reactivity were associated with the presence and severity of subclinical atherosclerosis in women with a history of PE.

Methods: Women aged 45-60 years with a 10 to 20 years earlier history of early onset preeclampsia (delivery <34 weeks of gestation) (n = 90), but without symptomatic CVD burden were screened for the presence of subclinical coronary artery disease (CAD) using both contrast-enhanced and non-contrast coronary CT angiography. Subclinical CAD was defined as a coronary artery calcium (CAC) score ≥100 Agatston Units and/or ≥50% coronary luminal stenosis. We assessed whether the numbers and activity of circulating neutrophils were associated with the presence of subclinical CAD and as secondary outcome measurements, with the presence of any calcium (CAC score > 0 AU) or stenosis, categorized as absent (0%), minimal to mild (>0 and <50%), and moderate to severe (≥50%) narrowing of the coronary artery. Blood was drawn just before CT and neutrophil numbers were assessed by flow cytometry. In addition, the presence of the chemokine receptors CXCR2 and CXCR4, which are known to be instrumental in neutrophil recruitment, and neutrophil activity upon stimulation with the bacterial peptide N-Formylmethionyl-leucyl-phenylalanine (fMLF) was assessed by flow cytometry.

Results: Of the participating women, with an average age of 49 years, 13% (12 out of 90) presented with subclinical signs of CAD (CAC score ≥100 AU and/or ≥50% luminal stenosis), and 37% (33 out of 90) had a positive CAC score (>0). Total white blood cell count and neutrophil counts were not associated with the presence of subclinical CAD or with a positive CAC score. When assessing the presence of the chemokine receptors CXCR4 and CXCR2, we observed a slight decrease of neutrophil CXCR2 expression in women with CAC (median MFI 22.0 [interquartile range (IQR) 20.2-23.8]) compared to women without CAC (23.8 [IQR 21.6-25.6], p = 0.02). We observed no differences regarding neutrophil CXCR4 expression. In addition, expression of the early activity marker CD35 was slightly lower on neutrophils of women with subclinical CAD (median MFI 1.6 [IQR 1.5-1.9] compared to 1.9 [IQR 1.7-2.1] in women without CAD, p = 0.02). However, for all findings, statistical significance disappeared after adjustment for multiple testing.

Conclusion: Our findings indicate that neutrophil counts and (re)activity are not directly associated with silent CAD disease burden and as such are not suitable as biomarkers to predict the presence of subclinical CAD in a high-risk population of women with a history of preeclampsia.

Keywords: coronary artery disease; neutrophils; preeclampsia; women.

Figure 1

Surface expression of neutrophil activity markers. Upon stimulation with N-Formylmethionyl-leucyl-phenylalanine (fMLF, 1 μM), surface expression of activity markers CD35, CD11B, and CD66B increases, whereas CD62L surface expression decreases (A). Moreover, the index values (after/before stimulation with fMLF) are shown (B). Data are presented as median with interquartile ranges from all patients (n = 90). *** indicates p < 0.001 (Mann–Whitney U test).

Figure 2

White blood cell (WBC), lymphocyte, monocyte, and neutrophil counts of the total study population stratified by presence of coronary artery calcification (CAC) (A), stenosis (B), and subclinical coronary artery disease (CAD) (C). Although the granulocyte count is slightly increased in former PE women with 50%–99% stenosis and subclinical CAD, these differences were not statistically significant. CAC indicates coronary artery calcification defined as >0 Agatston Units (AU); CAD, subclinical coronary artery disease defined as ≥100 AU and/or ≥50% stenosis.

Figure 3

Presence of chemokine receptors CXCR2 and CXCR4 is shown in women with a history of PE. The percentages of cells expressing CXCR2 or CXCR4 were comparable between women with or without presence of CAC, stenosis, and subclinical CAD (A–C). While the percentage of CXCR2 negative cells was comparable (A), CXCR2 surface expression was lower in women with CAC as compared to women without CAC (D). We observed no differences for CXCR2 or CXCR4 regarding the presence of stenosis or subclinical CAD (E,F). CAC indicates coronary artery calcification defined as >0 Agatston Units (AU); CAD, subclinical coronary artery disease defined as ≥100 AU and/or ≥50% stenosis. * indicates p < 0.05 (Mann–Whitney U test).

Figure 4

Neutrophil activity is shown in women with former PE. Neutrophil activity was not different between women with and without CAC (A). In women with >50% of coronary stenosis and with subclinical CAD, the neutrophil CD35 index was lower, indicating a lower response to stimulation with the chemotactic peptide fMLF (B,C). A similar trend was observed for degranulation marker CD11B, but not for CD66B and CD62L (B,C). CAC indicates coronary artery calcification defined as >0 Agatston Units (AU); CAD, subclinical coronary artery disease defined as ≥100 AU and/or ≥ 50% stenosis. * indicates p < 0.05 (Kruskal–Wallis test and Mann–Whitney U test).