Gestational diabetes mellitus modulates cholesterol homeostasis in human fetoplacental endothelium

https://doi.org/10.1016/j.bbalip.2018.05.005Get rights and content

Highlights

  • GDM causes increased ROS production and ROS-generated oxysterols in fetoplacental endothelial cells (HPEC).

  • Cholesterol efflux from HPEC is promoted in response to LXR activation.

  • LXR targets ABCA1 and ABCG1 are upregulated in GDM-HPEC along with increased cholesterol efflux via LXR activation.

  • De novo cholesterol biosynthesis is enhanced in GDM through upregulation of HMGCR.

  • Cholesterol homeostasis is adapted in HPEC under GDM condition.

Abstract

Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H2DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography–mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [3H]-cholesterol labeled HPEC and [14C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7β-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL3 (78 ± 17%, 40 ± 9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality.

Introduction

Gestational diabetes mellitus (GDM), defined as glucose intolerance first diagnosed in the second and third trimester, affects 6–13% of all pregnant women worldwide [1], 20–50% of whom progress to type-2 diabetes mellitus (T2DM) within 5–10 years [2]. Exposure to GDM in utero significantly increases the risk of obesity, T2DM, and cardiovascular diseases in offspring [3,4]. Such consolidated findings suggest that GDM modifies the metabolic programming of offspring early in development in utero.

The human placenta provides a selective physical barrier which is mainly responsible for exchange of maternal nutrients and fetal metabolites [5]. The human fetoplacental endothelium is in direct contact to the fetal circulation and is, thus, prone to be modified by metabolic products and hormones present in the fetal blood [6]. GDM results in altered metabolite concentrations in the fetal circulation such as hyperglycemia, hyperinsulinemia, and higher cholesterol levels and high-density lipoproteins (HDL)-triglycerides [7,8], which may modify the function of fetoplacental endothelial cells. GDM induces placental endoplasmic reticulum stress [9]. Moreover, GDM is associated with oxidative stress during pregnancy as a consequence of increased generation of reactive oxygen species (ROS) and/or suppression of anti-oxidative defense mechanisms [10]. ROS affect the functionality of the human placenta including hypervascularization and endothelial dysfunction while maternal diabetes contributes to abnormal fetal vascular function and impairs the fetal coronary artery vasculature [[11], [12], [13], [14], [15], [16]]. Diabetes-associated placental vascular dysfunction is widely considered as an early step in the pathogenesis of atherosclerosis [17]. Although pre-atherosclerotic lesions can be found in fetal aortas [18], pre-atherosclerotic lesions in the fetoplacental vasculature of the placenta have never been reported in GDM pregnancies. Thus, protective mechanism(s) against plaque formation may exist within the fetoplacental interface, potentially in endothelial cells.

Cholesterol is an essential component of every cellular membrane to maintain integrity and membrane-associated signaling cascades [19]. The accumulation of cholesterol in macrophages causes the formation of foam cells, which is an initial event in atherosclerosis [20]. However, endothelial cells in the feto-placental vasculature of the placenta proper have never been reported to show phenotypic changes similar to foam cells seen in macrophages, a change characterized by the unrestricted accumulation of cellular cholesterol [21]. Therefore, distinct and efficient mechanisms of regulating cholesterol homeostasis may be expected in vascular endothelial cells. One of the many known (athero)protective functions of plasma HDL is their central role in reverse cholesterol transport resulting in transfer of peripheral excess cellular cholesterol to mainly apolipoprotein (apo)A-I, thereby assembling to HDL, and releasing cholesterol to the liver for elimination [22]. We earlier defined mechanisms of effective cholesterol release from HPEC via two cholesterol transporters, ATP-binding cassette transporter (ABC)A1 and ABCG1 [23]. These encompass a two-step process involving ABCA1 and ABCG1 and their respective cholesterol acceptors, lipid-free apoA-I or apoE, and HDL, respectively [23]. In addition, we found phospholipid transfer protein (PLTP) expressed in HPEC. PLTP is involved in cholesterol transfer from HDL3 to HDL2 for subsequent clearance by the fetal liver and is upregulated in HPEC of GDM pregnancies [7]. Intriguingly, all three genes, i.e. ABCA1, ABCG1 and PLTP, are direct target genes for liver-X receptors (LXRs) that act as sterol sensors and regulate genes involved in cholesterol homeostasis, lipid and glucose metabolic pathways [24]. The endogenous ligands and activators of LXRs are oxysterols generated by either enzymatic catalysis or ROS-mediated oxidation [25]. Oxysterols are involved in many biological activities including regulation of cholesterol and steroid hormone biosynthesis, lipid homeostasis, inflammation, and cytotoxic effects [[26], [27], [28]]. Oxysterols are present at very low concentrations in the circulation of healthy humans [29]. Interestingly, elevated oxysterol levels have been detected in plasma of diabetes mellitus and atherosclerosis [30,31]. Fetal oxysterol levels have also been found elevated in pregnancies exposed to oxidative stress in a rat model of genetic disorders of fetal cholesterol biosynthesis [32]. The literature on oxysterols in the human fetal circulation is extremely limited [33] and little is known on the role of GDM in affecting the levels of fetal oxysterols.

Here we hypothesized that GDM increases oxidative stress and affects intracellular cholesterol metabolism in the fetoplacental endothelium. To test this, we applied a well-established in vitro model of primary HPEC isolated from control and GDM placentas [34] and investigated the effects of GDM on ROS formation, oxysterol levels, and key cholesterol metabolic processes involved in efflux, synthesis, and sequestration in these cells.

Section snippets

Subjects

The ethics committee of the Medical University of Graz approved this study (27-265 ex 14/15). All individuals gave voluntary informed consent and underwent an oral glucose tolerance test (OGTT) at 24 weeks of gestation. Control subjects were selected based on negative OGGT. Women with GDM diagnosed according to the WHO/IADPSG criteria [35], but without other pregnancy complications, were recruited before delivery. All subjects included in the GDM group were managed by diet and lifestyle

GDM induces ROS production and increases ROS-derived oxysterols in HPEC

To investigate whether GDM induces oxidative stress in primary HEPC, we first determined cellular ROS formation. We detected increased levels of H2DCFDA-reactive species formed during 24 h (1.3 ± 0.04-fold, p = 0.0001 Fig. 1A) in GDM versus control HPEC. This increase was attenuated (1.1 ± 0.04-fold, p = 0.0007 Fig. 1A) by incubating in presence of 100 μM Tiron, a potent scavenger of superoxide ions and free electrons [38]. These data suggest that GDM induces oxidative stress in HPEC by

Discussion

Our study demonstrates that HPEC isolated from GDM placentas significantly increase ROS generation as compared to control HPEC. In GDM, fetal levels of oxysterols formed by ROS and 27-OHC are elevated. Further, increased ROS-derived oxysterols lead to LXR activation in HPEC. GDM increases cholesterol efflux by upregulating ABCA1 and ABCG1, while the LXR antagonist GGPP reversed this upregulation entailing a reduction of the increased cholesterol efflux in GDM HPEC. Thus, GDM regulates

Conclusion

Collectively, our study demonstrates that GDM induces oxidative stress by increased ROS generation, and increased ROS-derived oxysterols may lead to LXR activation. GDM increases cholesterol efflux in response to LXR activation despite an increased cholesterol biosynthesis rate in HPEC. Human fetoplacental endothelium adapts its cholesterol homeostasis through ROS-generated oxysterols in GDM (Fig. 8). These findings provide new insights in protective mechanisms of the human placenta in

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Acknowledgements

We thank statistician BA. Katharina Eberhard (Medical University of Graz) for assisting statistical analysis and Prof. Kerry-Anne Rye (University of New South Wales) for valuable suggestions on the manuscript. This work was supported by the PhD program Molecular Medicine by the Medical University of Graz (C.W., G.D., U.P.), and the Austrian Science Fund, grants DK-MCD W1226-B18 (to U.P.) and P24783 (to U.P.).

Author contributions

Y.S. was responsible for the experimental design, carried out the experiments, performed data analysis, and wrote the manuscript. S.K., J.S., C.C.G., E.F-D., M.Z., S.C. R.S. and I.B. were involved in part of experiments and performed data analysis. A.K and S.F. provided know-how and materials for cellular ROS measurements. G.D., C.W. and U.P. were responsible for the experimental design and wrote/revised the manuscript. All authors read and approved this manuscript. Y.S., G.D., C.W. and U.P.

Conflict of interest statement

The authors declare no conflicts of interest.

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