Maternal Gestational Diabetes Mellitus increases placental and foetal lipoprotein-associated Phospholipase A2 which might exert protective functions against oxidative stress

Abstract

Increased Lipoprotein associated phospholipase A₂ (LpPLA₂) has been associated with inflammatory pathologies, including Type 2 Diabetes. Studies on LpPLA₂ and Gestational Diabetes Mellitus (GDM) are rare, and have focused mostly on maternal outcome. In the present study, we investigated whether LpPLA₂ activity on foetal lipoproteins is altered by maternal GDM and/or obesity (a major risk factor for GDM), thereby contributing to changes in lipoprotein functionality. We identified HDL as the major carrier of LpPLA₂ activity in the foetus, which is in contrast to adults. We observed marked expression of LpPLA₂ in placental macrophages (Hofbauer cells; HBCs) and found that LpPLA₂ activity in these cells was increased by insulin, leptin, and pro-inflammatory cytokines. These regulators were also increased in plasma of children born from GDM pregnancies. Our results suggest that insulin, leptin, and pro-inflammatory cytokines are positive regulators of LpPLA₂ activity in the foeto-placental unit. Of particular interest, functional assays using a specific LpPLA₂ inhibitor suggest that high-density lipoprotein (HDL)-associated LpPLA₂ exerts anti-oxidative, athero-protective functions on placental endothelium and foetus. Our results therefore raise the possibility that foetal HDL-associated LpPLA₂ might act as an anti-inflammatory enzyme improving vascular barrier function.

Figure 1

Overview of the study design and experimental set-up. The study investigated LpPLA₂ in three sample matrices: (1) isolated placental Hofbauer cells, (2) total placental tissue, and (3) on lipoproteins isolated from foetal cord blood plasma drawn immediately after delivery. Tissue and plasma were sampled from both Control and GDM subjects and used as described in the Material and Method section. Abbreviations: HBCs = Hofbauer cells; GDM = gestational diabetes mellitus; LpPLA₂ = lipoprotein-associated phospholipase A2; LDL = low density lipoprotein; HDL = high density lipoprotein; ELISAs = enzyme-linked immunosorbent assays; ECIS = electrical cell-substrate impedance sensing; DHR = 123-dihydrorhodamine.

Figure 2

LpPLA₂ is released by Hofbauer cells and increased in GDM. (a–c): Immunofluorescence staining of placental tissue. Images are representative of 3 independent experiments (N = 3). (a) van Willebrandt factor (vWF, green) was localized to the placental vessel lumen. LpPLA₂ (red) was localized to villous stroma and sub-endothelial connective tissue layers. EC = endothelial cells, SEL = sub-endothelial layer. (b) Trophoblast marker Cytokeratin 7 (CK7, green) was present in the fused syncytial layer of the villous; LpPLA₂ (red) was localized to villous stroma. SC = Syncytium. (c) Co-localization of LpPLA₂ (red) with CD163 (green), a marker of Hofbauer cells, was observed within villous stroma. HBC = Hofbauer cells. (d) LpPLA₂ activity secreted by HBCs isolated from healthy and GDM placenta (mean ± SD; N = 5 HBC isolations/group; two-way ANOVA). (e) LpPLA₂ activity is abolished by addition of 150 nM Darapladib, a specific LpPLA₂-inhibitor (mean ± SD, N = 4).

Figure 3

Metabolic hormones, cytokines and adhesion molecules modulate LpPLA₂ levels in Hofbauer cells. (a) Effect of Insulin on LpPLA₂ protein and activity in HBCs. (b) Effect of Leptin on LpPLA₂ protein and activity in HBCs. (c) Effect of TNFα (tumour necrosis factor α) on LpPLA₂ protein and activity in HBCs. (d) Effect of ICAM-1 (intra-cellular adhesion molecule 1; dashed line) and VCAM-1 (vascular adhesion molecule 1; solid line) on LpPLA₂ protein and activity in HBCs. (e) Effect of IL-4 (interleukin 4; dashed line) and IL-13 (interleukin 13; solid line) on LpPLA₂ protein and activity in HBCs. For each stimulus, at least four independent experiments were performed. Due to inter-individual variability of LpPLA₂ levels secreted, the unexposed control was used as baseline level and effects on LpPLA₂ activity where expressed relative to this control. p-values were calculated using one-way ANOVA.

Figure 4

LpPLA2 is present on foetal lipoproteins and increased in GDM. (a) Altered distribution of LpPLA₂ activity among lipoproteins of adult and neonate subjects. Lipoproteins were isolated from adult and cord blood plasma samples (N = 4, mean ± SD, one-way ANOVA). (b) LpPLA₂ activity of foetal lipoproteins from healthy and GDM pregnancies. Data from 3 individual experiments including a total of 21 subjects per group are shown. Statistical significance was calculated using one-way ANOVA. Abbreviations: GDM = gestational diabetes mellitus; LDL = low density lipoprotein; HDL = high density lipoprotein.

Figure 5

Hormones and Cytokines in neonatal circulation. Enzyme linked immunosorbent assays were used to measure (a) insulin, (b) leptin, (c) intracellular adhesion molecule 1 (ICAM-1), (d) vascular adhesion molecule 1 (VCAM-1) and (e) interleukin 4 (IL-4). Tumour necrosis factr α (TNFα) and interleukin 13 (IL-13) could not be quantified. All mean ± SD, students t-test.

Figure 6

Spearman Correlation of foetal LpPLA₂ activity with maternal BMI. (a) Correlation of LDL-LpPLA₂ activity in the foetus with maternal BMI. (b) Correlation between foetal LDL-LpPLA₂ activity with maternal BMI at delivery. (c) Correlation of HDL-LpPLA₂ activity in the fetus with maternal BMI before pregnancy. (d) Correlation of foetal HDL-LpPLA₂ activity with maternal BMI at delivery. (e) Correlation between foetal HDL-LpPLA₂ activity with maternal gestational weight gain. Abbreviations: BMI = body-mass index; LDL = low density lipoprotein; HDL = high density lipoprotein; LpPLA₂ = lipoprotein associated phospholipase A₂.

Figure 7

Surrogate markers of oxidative stress are inversely associated with LpPLA₂ in placenta and foetal circulation. (a and b) Western Blot of control and GDM placental tissue lysates against LpPLA₂ (a) and oxidized phospholipid residues (E06 oxPL) (b). ß-Actin was used for normalization as loading control. One representative out of N ≥ 3 experiments is shown. Images shown have been cropped; uncropped original files are available as Supplementary Information. (c and d) Densitometric analysis of LpPLA₂ protein and proteins modified by oxidized phospholipids (oxPL) in control and GDM placentae (N = 12 per group). Data from 3 individual experiments were pooled, mean ± SD, t-test. (e) Thiobarbituric acid reactive species (TBARS) were measured as surrogate of oxidative stress in neonatal cord plasma (mean ± SD, N = 16 per group, t-test). (f) Pearson correlation of TBARS levels in foetal circulation with HDL-LpPLA₂.

Figure 8

Anti-oxidative potential of HDL-LpPLA₂ on neonatal placental endothelium. (a) Barrier function assay of placental endothelial cells exposed to (i) oxidized phospholipid mix (oxPL, 15 ug/ml, green), (ii) oxPL plus neonatal HDL (15 ug/ml + 200 ug/ml, dark blue) and (iii) oxPL plus neonatal HDL in the presence of Darapladip (15 ug/ml + 200 ug/ml + 150 nM, resp.; turquoise). Darapladib alone (light blue) did not show any off-target effects compared to endothelial basal medium (EBM, red). One out of five representative experiments is shown. *p < 0.05 compared to EBM, †p < 0.005 compared to oxPL. (b) Anti-oxidative effects of HDL-LpPLA₂ in a cell-based assay of lipid peroxidation (ClickIT™ assay). Lipid peroxidation was visualized based on linoleamid alkyl and fluorophores on a laser scanning microscope using defined settings for all pictures taken to make them comparable. (c) Cell free assay of Control HDL (black) and GDM- HDL (grey) anti-oxidative potential based on the oxidation of 123-dihydrorhodamine (DHR). In addition to LpPLA₂-inhibition by Darapladib, also Paraoxanase-1 (PON-1) was inhibited by 2-hydroxyquinolone (2-OHQ). One-way ANOVA was used to test for significance.