Levels of Plasma Endothelin-1, Circulating Endothelial Cells, Endothelial Progenitor Cells, and Cytokines after Cardiopulmonary Bypass in Children with Congenital Heart Disease: Role of Endothelin-1 Regulation

Abstract

Congenital heart disease (CHD) can be complicated by pulmonary arterial hypertension (PAH). Cardiopulmonary bypass (CPB) for corrective surgery may cause endothelial dysfunction, involving endothelin-1 (ET-1), circulating endothelial cells (CECs), and endothelial progenitor cells (EPCs). These markers can gauge disease severity, but their levels in children's peripheral blood still lack consensus for prognostic value. The aim of our study was to investigate changes in ET-1, cytokines, and the absolute numbers (Ɲ) of CECs and EPCs in children 24 h before and 48 h after CPB surgery to identify high-risk patients of complications. A cohort of 56 children was included: 41 cases with CHD-PAH (22 with high pulmonary flow and 19 with low pulmonary flow) and 15 control cases. We observed that Ɲ-CECs increased in both CHD groups and that Ɲ-EPCs decreased in the immediate post-surgical period, and there was a strong negative correlation between ET-1 and CEC before surgery, along with significant changes in ET-1, IL8, IL6, and CEC levels. Our findings support the understanding of endothelial cell precursors' role in endogenous repair and contribute to knowledge about endothelial dysfunction in CHD.

Keywords: biomarkers; cardiopulmonary bypass surgery; children; circulating endothelial cells; congenital heart disease; cytokines; endothelial progenitor cells; endothelin-1.

Figure 1

Heatmap of corresponding Spearman correlation coefficients for the variables analyzed before and after surgery, where the positive correlation (directly proportional) is shown in blue and the negative correlation (inversely proportional) is shown in red, according to the color scale on the side of each subpanel. HPF corresponds to the group with high pulmonary flow, and LPF corresponds to the low pulmonary flow group. p values are presented in Table 2.

Figure 2

Comparative intergroup analysis of ET-1 plasma levels, CECs, and EPCs. Subpanel (a) shows the comparison of plasma ET-1 levels in the three groups of patients before and after surgery. Subpanel (b) shows the intergroup comparison as a violin graph with a logarithmic scale for ET-1. Subpanels (c,d) correspond to intergroup comparisons of the absolute numbers of CECs and EPCs, respectively, with a logarithmic scale, before and after surgery. Statistically significant: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (post hoc Bonferroni test). The fainter color and empty circles in each group represent measurements 24 h before surgery, while the more intense color and empty squares in each group indicated measurement 48 h after surgery. In all subpanels the medians in each group are represented by a thicker black line.

Figure 3

Flow cytometric detection of endothelial cells. Identification of EPCs by expression of CD46 and CD133 in representative plots from patient (n = 3), panel (a), and control (n = 7) panel (b). Most cells were also positive for VEGF (Figure S1). Panel (c): Quantitative analysis showed a greater proportion of endothelial cells was observed in CHD patients than in the control group.

Figure 4

Plasma cytokine levels measured via flow cytometry in patients and controls. Panel (a) shows the comparison of plasma levels of IL-8 and IL-6 cytokines measured using flow cytometry in the three groups of patients, where it can be seen that the highest values of both cytokines were obtained in CHD LPF group. Panel (b) shows the comparison of plasma levels of IL-10 and IL12p70 in the three groups of patients before and after surgery; it can be seen that the highest levels of IL-10 are in the control group, followed by the CHD LPF group after surgery. Statistically significant: *** p ≤ 0.001.