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EX VIVO AND IN VITRO MODELS TO STUDY THE EFFECTS OF HYPOXIA AND INFLAMMATION ON HUMAN PLACENTAL MITOCHONDRIA
Full text linkIntrauterine Growth Restriction (IUGR) is a pregnancy-related pathology characterized by a placental insufficiency phenotype and a multifactorial etiology that still needs to be completely clarified. IUGR is associated with increased risk of maternal and neonatal perinatal mortality and morbidity and a tendency to develop cardiovascular and metabolic pathologies in the adulthood. A deeper knowledge of the alterations occurring in IUGR has therefore become essential to find therapeutic tools to prevent fetal, neonatal and future adult complications.
A specific placental phenotype has been associated with IUGR, characterized by placentation defects, altered transport of oxygen and nutrients to the fetus, impaired mitochondria content and increased oxidative stress (OxS).
Mitochondria (mt) are eukaryotic ubiquitous organelles whose number range from hundreds to thousands of copies per cell. As they are the fuel stations of all cells, more than 95% of ATP is synthesized in these organelles Besides this well-known function, many essential pathways involve mitochondria, such as mt biogenesis. Mt biogenesis is a complex of mechanisms needed to mitochondria ex-novo creation: mt DNA duplication and translation of mt factors controlling the transcription machinery that produce all respiratory chain complexes (RCC). IUGR hypoxic features, and the consequent higher OxS, affect mitochondria as showed by in vivo models increased mt oxygen consumption trigger by hypoxia or in vitro downregulation of mt biogenesis.
The aim of this study was to investigate, by ex vivo experiments and in vitro models, different types of placental cells to deeper characterize the placental insufficiency features of IUGR, with specific attention to the consequences of its hypoxic environment.
IUGR and physiological placenta bioenergetics were first examined, by analyzing both mitochondrial (mt) content and function in whole placental tissue and in several placental cell types (cytotrophoblast and mesenchymal stromal cells).
Mt DNA content resulted higher in IUGR placentas compared to controls, as well as NRF1 (biogenesis activator) mRNA levels. Oppositely, both mtDNA and NRF1 expression levels were significantly lower in cytotrophoblast cells isolated from IUGR placentas compared to controls. The observed divergence between placental tissue and cytotrophoblast cells may suggest that other placental cell types (e.g. syncytiotrophoblast, endothelial cells and mesenchymal stromal cells), that are subjected to different oxygen - and consequently oxidative stress - levels may be responsible for the mt content increase in the whole placental tissue. Moreover, a different exposure to progesterone may also explain this mt content divergence, since progesterone, regulating mt biogenesis, is produced by syncytio but not in cytotrophoblast cells.
In IUGR cytotrophoblast cells, respiratory chain complexes (RCC) showed lower, though not significantly, gene expression levels and no differences in their protein expression compared to controls. In contrast, mt bioenergetics - represented by cellular O2 consumption - was higher in IUGR versus controls, especially in more severe IUGR cases. Thus, despite the protein content of RCC was not altered, their activity was significantly increased in IUGR cytotrophoblast cells, possibly due to a more efficient RCC assembly. Finally, as O2 consumption resulted inversely correlated to mtDNA in cytotrophoblast cells, a functional (respiration) compensatory effect to the decreased mitochondrial content might be hypothesized.
Estrogen-Related Receptor (ERRγ) is a very interesting transcriptional factor involved both in mt biogenesis and function and in estradiol production (through CYP19 aromatase up-regulation). ERRγ and CYP19 mRNA levels were therefore analyzed, for the first time in human IUGR placentas.
In whole placental tissue CYP19 showed higher expression in IUGR compared to controls, progressively increasing with IUGR severity. Higher ERRγ expression in IUGR cases was also found, though not significantly. These data are consistent with mtDNA and NRF1 results, thus confirming altered mt biogenesis and content in IUGR and strengthening the hypothesis of a restore attempt made through the stimulation of mt biogenesis. An additional effect of ERRγ increase is CYP19 upregulation. The observed higher CYP19 expression may indicate a protective mechanism exerted through estradiol against oxidative stress.
Opposite to their placental tissue expression, ERRγ levels in cytotrophoblast cells significantly decreased in the IUGR group compared to controls. This is consistent with literature evidences of O2-dependent ERRγ gene expression in trophoblast cells. As well as for mt DNA and NRF1 levels, other cell types could be responsible for ERRγ increase in the whole placental tissue. CYP19 expression was not significantly different between IUGR and controls in cytotrophoblast cells, though it positively correlates with ERRγ levels, but low CYP19 levels are reported for cytotrophoblast cells, and this might complicate the detection of any difference. Interestingly, a significant positive correlation linked maternal BMI and expression of both ERRγ and CYP19 genes (in whole placental tissue: positive trend/cytotrophoblast cells: negative trend). An estradiol-dependent regulation of leptin production through ER (Estrogen Receptor) – ERR is known. Leptin, an anti-obesity hormone produced also by placenta, increase during. The future measure of plasmatic levels of both leptin and 17β estradiol in maternal blood will verify this speculation.
Then in vitro experiments were performed to assess possible biomolecular mechanisms regulating mithocondrial content in Intrauterine Growth Restriction, by culturing primary placental cells under normal oxygen conditions and hypoxia, a typical feature of IUGR. Fluctuations in placental oxygen concentration may generate oxidative stress (OxS), that is enhanced in Intrauterine Growth Restriction condition. As mitochondria are the major producers of intracellular reactive oxygen (O2) species through free radicals generated by the mt oxidative phosphorylation, altered intrauterine O2 conditions might affect mt DNA content and function, leading to increased oxidative stress in IUGR placental cells. Using trophoblast primary cell lines could help to understand O2 conditions that placentas may be exposed to in IUGR pregnancy. Exposure of trophoblast cultures to hypoxia is an in vitro model commonly used in the last few years. Preliminary data from performed experiments show that the oxygen lack in cytotrophoblast cells leads to increased mt DNA levels. The evidence that O2 levels may regulate mt biogenesis in cytotrophoblast cells highlights their deep sensitivity to O2 conditions. However, further data are needed to confirm these preliminary results, also considering the implied difficulties in adapting the primary cytotrophoblast cultures, very sensitive to O2 concentration, to an in vitro model. A future goal will be reproduce particularly hypoxia/re-oxygenation intervals characterizing placental insufficiency and generating OxS and measuring cell apoptosis levels and autophagy markers (e.g. TNF-α, p53, caspases).
Finally, in vitro experiments were performed to isolate and characterized p-MSCs from physiological and affected by IUGR placentas. p-MSCs have never been investigated before in IUGR pregnancies, but their role have been recently studied in preeclamptic placentas. PE p-MSCs show pro-inflammatory and anti-angiogenic features, that may result in abnormal placental development. In the performed p-MSCs cultures, mesenchymal markers enrichment and multipotent differentiation abilities confirm the successful isolation and selection of a mesenchymal stromal cell from placental membranes and basal disc of both physiological and IUGR placentas. As attested by flow cytometry data, the p-MSC population is earlier selected in IUGR placentas: this faster selection might represent a compensatory mechanism to metabolic alterations occurring in IUGR placental cells and/or to the adverse IUGR placental environment. During placenta development, the lower proliferation rate characterizing IUGR pMSCs could impair the primary villi formation and consequently trophoblast development, since MSCs both serve as structural of trophoblast cells.
Moreover, IUGR p-MSCs population display lower endothelial and higher adipogenic differentiation potentials compared to controls. During pregnancy, pMSCs usually contribute to both vasculogenesis and angiogenesis Interestingly, several studies report some alterations in maternal and fetal endothelial progenitor or in the angiogenic capacity of IUGR placental cells. Opposite to endothelial differentiation ability, the adipogenic potential in pMSCs from IUGR is increased compared to controls: as these changes are evident early in life, the predisposition to obesity may be programmed in utero. To further characterize IUGR pMSCs, their mitochondrial (mt) content was investigated by measuring NRF1 and Respiratory Chain UQCRC1 and COX4I1 gene expression levels. Mesenchymal stem cell metabolism is known to be mainly anaerobic, with a shift towards an aerobic mitochondrial metabolism reported during differentiation. Interestingly, p-MSCs cultured with no differentiating medium present a trend towards higher NRF1, UQCRC1 and COX4I1 expression levels in IUGR basal disc samples compared to controls and higher COX4I1 levels in IUGR placental membranes; these differences are not statistically significant likely because of the low sample number. Nevertheless, they might account for metabolic alterations in IUGR p-MSCs, showing a possible shift to aerobic metabolism, with the loss of the metabolic characteristics that are typical of multipotent and undifferentiated cells.
The different gestational age between cases and controls, typical of all IUGR versus term-placentas studies, is a possible limit that associate all the performed experiments. However, any significant correlation between gestational age (ge) and the O2 consumption of CIV (which presents the highest significance between IUGR and controls), ge and mt DNA levels, ge and ERRy/CYP19 expression, ge and p-MSCs. CYP19 gene expression have been analyzed assuming that it may represent an index of aromatase content in placental tissue. However, post-translational modifications (glycosylation and phosphorylation) may occur, affecting its functional activity. Finally, a potential limitation of placental mesenchymal stromal cells is that the analysis was performed on IUGR placentas at delivery, whereas placental abnormal development of IUGR pathology is supposed to start already at the beginning of placentation.
Taken together, reported data highlight mitochondrial alterations occurring in placentas of Intrauterine Growth Restricted pregnancies, through ex vivo and in vitro approaches.
These results shed genuine new data into the complex physiology of placental oxygenation in IUGR fetuses. Mitochondrial content is higher in IUGR total placental tissue compared with normal pregnancies at term. This difference is reversed in cytotrophoblast cells of IUGR fetuses, which instead present higher mitochondrial functionality. These findings suggest different mitochondrial features depending on the placental cell lineage.
Indeed, our results on placental Mesenchymal Stromal Cells, showed higher levels of genes accounting for mitohcondrial content and function. The increased placental O2 consumption by placental tissue may represent a limiting step in fetal growth restriction, preventing adequate O2 delivery to the fetus. This limitation has potential consequences on fetal O2 consumption both in animal models and in human IUGR
Is the placenta an innocent bystander in perinatal programming?
No full textDuring pregnancy, a well functioning placenta is needed to ensure appropriate growth and development of the fetus [1]. Indeed, a malfunctioning or “insufficient” placenta has been recognized as the “cause” of Intrauterine Growth Restriction (IUGR) [2], leading to decreased oxygen delivery as well as altered placental transport of nutrients, mainly amino acids and lipids, but also micronutrients such as iron and folate. A number of previous studies from our lab support this hypothesis, demonstrating a specific placental phenotype of IUGR [3], recently confirmed with decreased levels of placental Transferrin Receptor (TFRC – mediating cellular iron uptake) or of Sodium-coupled Neutral Amino acid transporter 2 (SNAT2) in IUGR versus controls [4, 5] (summarized in Tab. 1). Maternal nutritional status, diet and exposure to environmental factors are increasingly acknowledged as potentially affecting placental gene expression, thus modifying placental function. These epigenetic associations link intrauterine environment to adverse perinatal outcomes reprogramming the fetal epigenome with several mechanisms, such as methylation or miRNA, thus affecting gene expression and activity in preeclamptic (PE) and IUGR tissues [6]. Changes in miRNA expression pattern have been observed in placental tissue and associated with several pregnancy pathologies as preeclampsia (DOWN miR-21, UP miR-155, DOWN miR-223), GDM (DOWN miR-132), IUGR (DOWN miR-21, DOWN miR-210) and preterm birth (UP miR-493, UP miR-338) [7]. In this context, an active placental metabolism is crucial to support both trophoblast invasion and placentation [8]. Alterations in early implantation may lead to mismatches in oxygen (O2) delivery to different areas of the placenta, with less O2 exchange between the uterine and the umbilical circulations [9]. Mitochondrial DNA (mtDNA) copy number is positively correlated with the number of mitochondria. We have previously demonstrated altered mitochondrial content in IUGR placentas [10], with higher mtDNA levels in IUGR maternal blood [11]. Moreover, we measured the functionality of the respiratory complexes (RCC) by high-resolution respirometry (HRR), in order to assess potential alterations in placental energy metabolism [12] (summarized in Tab. 1). Preliminary observations suggest similar changes in placental mitochondria, DNA content and function of obese pregnant women. These pregnancies are characterized by low-grade inflammation and oxidative stress [13]. Moreover, dysregulated mt genes methylation (D-loop and CO1 hypomethylation) might expand our findings of higher mtDNA content in fetal cord blood of
IUGR and PE [14]. These preliminary data may indeed suggest a compensatory attempt of fetuses to increase energy production through higher mtDNA content and RCC (CO1) expression, representing a further link between epigenetic
changes and perinatal programming of diseases. Another issue is related to the placental hormonal function. The placenta as a source of a wide array of hypothalamic or pituitary hormones was a hot topic in the 60-70s, then neglected because of the radioactive techniques needed at that time. Steroid hormones, and in particular estrogens, are important for uterine/placental vascular adaptations to pregnancy, but also essential for trophoblast cells syncytialization in placenta. During pregnancy, the feto-placental unit is a source of estrogens through its aromatase enzyme Cytochrome P450 (CYP19) involved in estradiol (E2) production [15]. Interestingly, CYP19 levels appeared signi%cantly higher in IUGR placentas that we recently analyzed. We might speculate that the CYP19 alterations have an estrogenrelated protective action in more severe IUGR placentas, which we showed to be characterized by increased mtDNA [16]. Ongoing analyses will evaluate if these placental molecular alterations result in E2 hormone altered production. Placental mesenchymal stromal cells (p-MSCs) may also represent an interesting point to evaluate in order to understand normal and abnormal placental development. In IUGR pregnancies, p-MSCs have lower proliferation rate with earlier shift towards homogeneity than in controls. In vitro findings also demonstrate that multipotency of IUGR derived p-MSCs is restricted, as their capacity for adipocyte differentiation is increased, whereas their differentiation ability towards endothelial cell lineage is decreased (Fig. 1) [17]. These findings are indicative of changes that may also be reflected in the developing fetus (summarized in Tab. 1).
The potential role for p-MSCs in pregnancy pathologies, as well as the striking mitochondrial changes involved in energy production, open new perspectives for understanding the development of the diseases and potential routes of prevention and treatment
Ferroportin gene and protein expression in human IUGR placentas
No full textIron (Fe) deficiency in pregnancy is associated with low birth weight and premature delivery.
We demonstrated a significant decrease of the Fe cell-importer Transferrin Receptor (TfR1) in human Intrauterine Growth Restricted (IUGR) vs normal (N) placentas.
Ferroportin (FPN), located in the trophoblast cell (TC) basal membrane, exports Fe towards the fetal circulation. We hypothesized that TfR1 downregulation in IUGR placentas could be due to Fe intracellular accumulation, and we measured FPN expression in human IUGR vs N placentas.
Placentas were sampled at the time of elective cesarean section; villi were selected, washed and immediately frozen for following analysis. IUGR was defined by a reduction of more than 40 centiles in the growth of the abdominal circumference measured by ultrasound in utero and birth weight <10th percentile. Three severity groups were identified depending on the umbilical artery pulsatility index and fetal heart rate. FPN mRNA was quantified in 41 IUGR and 50 N placentas by Real Time PCR. Among them, we measured FPN protein expression in 14 IUGR and 26 N placentas, setting up a specific Enzyme-Linked ImmunoSorbent Assay (Elisa) protocol.
Both FPN mRNA and protein expression were not statistically different in IUGR vs N placentas, independently from the degree of severity.
Our results show no differences in IUGR and N FPN mRNA and protein placental levels. This suggests that Fe is not accumulated in IUGR TC, and the Fe reaching IUGR fetuses could be decreased compared to normal pregnancies, as a consequence of TfR1 downregulation in the microvillous membranes.
Since FPN is known to be post-transcriptionally finely regulated, we aim at enlarging our Elisa analysis case study in order to confirm our data, and then measuring Fe levels in the cord blood to verify our hypothesis
Hypoxia and mitochondrial DNA : primary human trophoblast culture as a tool to investigate mechanisms behind placental insufficiency pathologies
No full textThe placenta is an active organ between mother and fetus. Therefore it is essential to maintain the homeostasis for the developing fetus environment: adverse influences during intrauterine life may contribute to develop adult diseases, according to the fetal programming theory (Barker, MolMedToday 1995). Defects in placentation may lead to obstetric pathologies like Intrauterine Growth Restriction (IUGR) and Preeclampsia (PE), both associated with placental insufficiency (Levy et al, Am J Obstet Gynecol. 2002). PE and IUGR arise in a low oxygen microenvironment, while placenta hypoperfusion and hypoxia, occurring later in pregnancy, induce trophoblast injury (Oh et al, Placenta 2011).
Mitochondria (mt), key players of cellular respiration, may act as a possible link between oxygenation defects of placental cells and these pregnancy complications. Our group previously demonstrated significantly higher mitochondrial DNA content (mtDNA) in IUGR placentas versus controls (Lattuada et al, Placenta 2008). Moreover, a correlation between PE placentas and mitochondrial dysfunction has already been demonstrated and linked to oxidative stress (Myatt, Histochem Cell Biol. 2004; Hung and Burton, Taiwan J Obstet Gynecol. 2006). Other studies have shown the influence of acute and chronic hypoxia on mtDNA content in murine models (Widshwendter, MolMedToday 1998; Gutsaeva et al, J.Neurosci. 2008). These data highlight the necessity to better understand the oxygenation pathways in placental physiological cells and the effect of low oxygen in this system.
Our proposal is thus to study primary physiological trophoblast cells, an in vitro model already used in other studies, coltured under different oxygen conditions, and investigating the effects on mitochondria copy number.
Primary trophoblast cells were isolated as previously described (Kliman et al, Endocrinology 1986) from human placentas of term singleton uncomplicated pregnancies (non-smoking women with appropriately grown fetuses and no maternal/fetal pathologies). Trophoblasts were maintained in standard conditions (20% oxygen -O2-; 5% CO2; 37C°). After 4 hours (T0) cells were incubated in the following conditions: 20%O2; 20%O2 with 0,2mM Cobalt Chloride (CoCl2, activator of HIF1α) in the medium; 8%O2 (which mimics O2 conditions of 2nd-3rd trimester placentas); 0.1%O2. Cells were coltured for 72 hours and freezed at T24, T48, T72. A further condition was represented by shifting cells from 20% to 8 or 0.1% of O2 at T48, to investigate low O2 effects on already syncytialized cells. Total DNA was extracted by Trizol from cells freezed at T24, T48, T72. mtDNA was analyzed by real-time PCR (2-Ct method) using Cytocrome B as mt target gene and the nuclear RNasi P as endogenous gene.
At T24, T48, T72, all cells coltured at low O2 (8-0.1%) present a trend, though not significant, towards higher mtDNA levels compared to standard conditions, confirming a possible role of low O2 levels in up-regulating mtDNA levels. The same tendency is observed in cells shifted to 8 or 0.1% O2 at T48, thus showing a similar response in syncytiotrophoblast cells, and in 20%O2-CoCl2 conditions, possibly indicating a mediation by HIF1α. Data clustered by O2 concentration show no significant variations between different time intervals. These are preliminary data: we intend to increase the sample number and to reduce the variability in the experimental procedure. Moreover, we propose to standardize the number of cells plated and to extend the adhesion interval from 4 to 12 hours, improving the amount and quality of mtDNA. To enrich our data, we want to study further aspects of mt functionality, for example the possible alterations of respiratory chain complexes caused by lack of O2
Beta Estradiol Levels in Growth-Restricted Pregnancies
No full textIntroduction: Estrogen-Related Receptor gamma (ERRy) is an
activator of mitochondrial (mt) biogenesis. Moreover, ERRy can
affect the estrogen pathway by binding the Estrogen Receptor (ER)
or modulating CYP19 expression, an aromatase involved in 17-
17Beta-Estradiol (E2) production. During pregnancy, the fetus-placenta
unit is the primary source of estrogens. E2 is critical for placental
function and fetal growth.Werecently observed reduced ERRy and
mtDNA levels in trophoblast cells from growth-restricted (IUGR)
placentas. To evaluate if these placental molecular alterations could
affect 17-Beta Estradiol production, we measured E2 concentration
in maternal plasma of the same IUGR pregnancies.
Materials and Methods: Maternal venous blood collected
before cesarean section (no labor) from 11 IUGR and 16 term normal
pregnancies (Controls), centrifuged at 1500rpm x15 min. at
R.T. and obtained plasma stored at -80 ◦C.IUGRclassified in2groups
of increasing severity based on umbilical artery Pulsatility Index
(PI) and Fetal Heart Rate (FHR). E2 levels measured with an electrochemiluminescence
immunoassay (Elecsys Estradiol III- Cobas) and
an automated Elecsys immunoanalyser. Clinical and molecular data
analyzed with T-test and Pearson correlation (p < 0.05-SPSSv23.00).
Results: Maternal age and BMI did not differ between IUGR and
controls, while gestational age, placental and fetal weight, placental
area and fetus/placenta ratio were significantly lower in IUGR
(p≤0.001). 17-B Estradiol in maternal venous plasma resulted
significantly lower in IUGR (14893.0±6746.6 pg/mL) versus
controls (25351.2±10755.3 pg/mL)(p = 0.009)(Fig. 1). Analyzing
IUGR cases depending on severity, E2 concentration was lower in
both IUGR groups versus controls, reaching statistical significance
in those with normal PI (15305.4±7211.1 pg/mL,n = 7-p = 0.036;
abnormal PI:14171.2±6828,7 pg/mL, n = 4-p = 0.06). Maternal
plasma E2 levels positively correlated with gestational age,
fetal weight and placental area (p = 0.006,R = 0.516;p = 0.002,R
= 0.570;p = 0.015,R = 0.489). A strong positive correlation linked
plasma E2 with fetus-placenta ratio, an index of placental efficiency
(p = 0.006,R = 0.512)(Fig. 2). Conclusions:in extra-placental
tissues, 17-B Estradiol exerts a protective role against oxidative
stress (OxS). This is mediated by ERalpha and its co-activator ERRy,
possibly inducing a reduction of mt OxS. We recently showed that
ERRy, which binds CYP19 promoter, was downregulated in IUGR
trophoblast cells, where 17-B Estradiol is produced. The significant
difference found in maternal plasma 17-B Estradiol may therefore
confirm an altered status in IUGR placentas. These present
reduced surface and efficiency and interestingly both parameters
here correlate with E2 concentration. These data support the
hypothesis that placental insufficiency may be characterized
by an ERRy-mediated impairment in placental steroidogenesis
that increases IUGR susceptibility to the oxidative intrauterine
environment.Supported by FGP/MIUR
Sex specific adaptations in placental biometry of overweight and obese women
No full textIntroduction: Placental biometry at birth has been shown to predict chronic disease in later life. We hypothesized that maternal overweight/obesity, a state of low-grade inflammation and risk factor for adverse pregnancy outcome, could negatively influence placental development and that differences would be sex-specific.
Methods: 696 women (537 normal-weight, NW; 112 overweight, OW; 47 obese, OB) with singleton uncomplicated pregnancies were prospectively enrolled at term delivery. Gestational age, maternal (age, height, pre-pregnancy BMI, gestational weight gain -GWG, hemoglobin, hematocrit and glycemia), fetal (weight, length, ponderal index, cranial circumference) and placental (weight, diameters) data were collected. Placental area, thickness and efficiency (fetal/placental weight ratio, F/P) were calculated. Results: GWG was within standard recommendations in OB, while OW exceeded it. Placental weight was significantly higher in OW versus NW, but not in OB, leading to significantly higher placental thickness and lower F/P in this group.
In the total population, a significant interaction effect between maternal BMI and fetal sex on placental weight and efficiency was found. Indeed, differences in placental parameters were present only in female offspring.
Discussion: In our population of OW and OB uncomplicated pregnancies only OW women, presenting GWG over standard recommendations, had thicker and less efficient placentas. We also reported different placental adaptation depending on fetal sex, with significant changes only in female fetuses. This may be part of a female-specific strategy aiming to ensure survival if another adverse event occurs. Customized counseling according to maternal BMI and fetal sex should be evaluated in clinical care
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