The 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.

Hypoxia and mitochondrial DNA : primary human trophoblast culture as a tool to investigate mechanisms behind placental insufficiency pathologies / G.M. Anelli, C. Mandò, M.A. Marino, M. Figus, I. Cetin. ((Intervento presentato al 15. convegno Agorà SIMP : il luogo del libero scambio delle idee e degli argomenti tenutosi a Genova nel 2012.

Hypoxia and mitochondrial DNA : primary human trophoblast culture as a tool to investigate mechanisms behind placental insufficiency pathologies

G.M. Anelli
Primo
;
C. Mandò
Secondo
;
M.A. Marino;M. Figus
Penultimo
;
I. Cetin
Ultimo
2012

Abstract

The 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.
23-nov-2012
hypoxia ; mitochondrial DNA ; trophoblast culture ; placental insufficiency pathologies
Settore MED/40 - Ginecologia e Ostetricia
Società Italiana di Medicina Perinatale (SIMP)
Hypoxia and mitochondrial DNA : primary human trophoblast culture as a tool to investigate mechanisms behind placental insufficiency pathologies / G.M. Anelli, C. Mandò, M.A. Marino, M. Figus, I. Cetin. ((Intervento presentato al 15. convegno Agorà SIMP : il luogo del libero scambio delle idee e degli argomenti tenutosi a Genova nel 2012.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/214173
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