Acute sensitivity of first trimester placental tissue to oxygen tension in vitro
1
A.28 Hypoxia upregulates the tmnscription of vascular endothelial growth factor and its receptor in tmphoblast cell lines. C.M. Tavlor, F.W. Anthony & T. Wheeler, Obstetrics and Gynaecology, Faculty of Medicine, University of Southampton, UK. We investigated the influence of hypoxia on the trophoblast cell lines (JEG, JAR & BeWo). The cells were cultured to 80% confluence and thereafter incubated for 24 hours in either normoxia or hypoxia (achieved by sealing the culture plates in airtight bags with a palladium catalyst to absorb oxygen). Oxygen tension in the culture media was 26 kPa at the start of the experiments; thereafter it fell and remained at 19 kPa (normoxia) or 5 kPa (hypoxia). RNA was extracted after 24 hours and reverse transcription and competitive PCR used to examine the expression of the alternatively spliced forms of vascular endothelial growth factor (VEGF) and the transmembrane region of the VEGF receptor, flt-1 . VEGF and its receptor were upregulated in all 3 cell lines in the hypoxic cultures when compared with the normoxic. VEGF protein was also measured and produced in greater amounts (per cell) in the hypoxic cultures. The regulation of VEGF production through hypoxia may have a direct influence on trophoblast invasion particularly in early pregnancy when local oxygen tension is low. VEGF may effect this by increasingprotease production and providing a directional stimulus for trophoblast cell movement through local oxygen gradients. Placenta (1996), Vol. 17 Acute sensitivity of first trimester placental tissue to oxygen tension in vitro. Adrian Watson, Marion Palmer, Eric Jauniaux and Graham Burton, Department of Anatomy, University of Cambridge and University College Hospital, London, UK. When maintaining first trimester human placental villi in organ culture we observed rapid degeneration of the syncytium within 24 hours, despite excellent viability of the cytotrophoblastic and stromal cell types. It was discovered that after handling under ambient conditions mitochondrial activity had all but ceasedwithin the syncytium by 2 hours post-termination. This situation could only be affected by using tissue of 14 weeks-plus gestational age, when the syncjXium showed muih improved survival and mitochondrial activitv. The sueed of the effect on i I mitochondria led us to consider that oxidative mechanisms may be the cause of syncytial death. We therefore attempted to maintain villi under low oxygen conditions (2.5%) rather than ambient levels (2 1%). We found that syncytial survival was reproducibly improved and mitochondrial activity retained at time zero levels for 6 hours and more. This effect could only be guaranteed if the tissue was placed into the low oxygen environment within a very short time following removal from the uterus. Furthermore, we observed that neither the copper/zinc nor the manganese forms of superoxide dismutase were present in detectable quantities within the syncytium of 8 and 9 week tissue and only in certain areas of lo-12 week tissue. However, 14-16 week tissue showed considerable amounts of Cu/Zn SOD in the svncvtiotroohoblast. At all sees (8-16 weeks) Cu/Zn SOD ias ietecte‘d in the cytotrophvobl&ts and the ‘stromal cells. These results are in agreement with the proposal that the first trimester of gestation occurs under a low oxygen tension. Supported by the Wellcome Trust (03805 l/Z/93/Z/l S). CONTROL OF OXYGEN DELIVERY IN DUAL PERFUSION OF AN ISOLATED HUMAN PLACENTAL LOBULE. B. M. Polliotti, AS. Bukhtiar, L. Jessee, C. van den Revdt. D. Comish and R.K. Miller - Deots of Obstetrics and Gyhecology and Environmental Medicine, University of Rochester Medical Center, NY, & *Dept of Pediatrics, Emory University, Atlanta, Georgia, USA. The efficiency of oxygenation in the human placenta under dual perfusion conditions has always been an important issue in studying normal placental function as well as hypoxia and toxicity. The aim of this study was to investigate improvements in the control of oxygen delivery to the dually perfused isolated human cotyledon using a membrane oxygenator designed for cardiopulmonary bypass applications. A A&a Silo.+S 0.3 membrane oxygenator (Senko Medical Instruments, Tokyo, Japan) replaces -traditional oxygenation systems (bubbling, rotation, etc.) used to deliver gases to both the maternal and fetal perfusate. Perfusate consists of Ml99 tissue culture medium supplemented with dextran, glucose, L-glutamine, sodium bicarbonate, heparin and antibiotics. Intensive measurements have been done using different concentrations of O2 (0% to 95% 02, with 5% Cd,, balanced with N7). Maternal oerfusate can be 0, “loaded” from 70 to 650 -, ‘. mmHg in a strictly linear manner by using this membrane oxygenator with different concentrations of 02 in the gas mixture. Without any oxygen carrier (red blood cells, cytochromes or flurocarbons), this membrane oxygenator allows a very precise control of the oxygen provided to the maternal circuit. Studies requiring hypoxia conditions can be achieved more easily with this device. In addition, this oxygenation system protects the compound (e.g., proteins) added in the perfusate from being degraded or modified by the gases. (Supported in part by NIH Grants - ES 02774 and ES 01247). The Effects of A4aternal Smoking on Placental Oxygell Transfer. P.G.Bush’, T.M.Mayhew2, D.R.Abramovich3, M.D.Burke’, P.J.Aggett’, & K.R.Page’. Departments of ‘Biomedical Sciences, and 30bstetrics and Gynaecology, Aberdeen University, Aberdeen, 2Human Morphology, Nottingham University, Nottingham, and “Pharmaceutical Sciences, De Montford University, Leicester, ‘Institute of Food Research, Norwich, United Kingdom. Maternal smoking may have an adverse effect on the transport of respiratory gases to the fetus via changes in placental morphology. In this study,placentas were obtained at delivery from healthy uncomplicated pregnancies and grouped according to maternal smoking habit. Random tissue samples were obtained and following fixation and section staining, structural quantities were estimated and used to calculatethe total diffusive conductance for oxygen Dp (Mayhew et al. 1990). Significant morphological changes were observed (including the arithmetic mean of stromal thickness which increased).However, there was no significant change in Dp (cm3 ) 0,. mid’. kPd’) mean (SEM): 17.3 (2.2) (n=ll) in non-smokers and 15.0 (0.8) (n=27) in smokers. Taking into account fetal weight we found also no difference between speicific difisive conductances. Thus despite changes in placental morphology the apparent 13% decline in oxygen diffusive conductance did not attain statistical significance. (Supported by the Tobacco Products Research Trust). Reference: Mayhew TM, Jackson MR, & Haas JD (1990) Placenta 11.493-503.
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Transcript of Acute sensitivity of first trimester placental tissue to oxygen tension in vitro
A.28
Hypoxia upregulates the tmnscription of vascular endothelial growth factor and its receptor in tmphoblast cell lines. C.M. Tavlor, F.W. Anthony & T. Wheeler, Obstetrics and Gynaecology, Faculty of Medicine, University of Southampton, UK.
We investigated the influence of hypoxia on the trophoblast cell lines (JEG, JAR & BeWo). The cells were cultured to 80% confluence and thereafter incubated for 24 hours in either normoxia or hypoxia (achieved by sealing the culture plates in airtight bags with a palladium catalyst to absorb oxygen). Oxygen tension in the culture media was 26 kPa at the start of the experiments; thereafter it fell and remained at 19 kPa (normoxia) or 5 kPa (hypoxia). RNA was extracted after 24 hours and reverse transcription and competitive PCR used to examine the expression of the alternatively spliced forms of vascular endothelial growth factor (VEGF) and the transmembrane region of the VEGF receptor, flt-1 . VEGF and its receptor were upregulated in all 3 cell lines in the hypoxic cultures when compared with the normoxic. VEGF protein was also measured and produced in greater amounts (per cell) in the hypoxic cultures. The regulation of VEGF production through hypoxia may have a direct influence on trophoblast invasion particularly in early pregnancy when local oxygen tension is low. VEGF may effect this by increasing protease production and providing a directional stimulus for trophoblast cell movement through local oxygen gradients.
Placenta (1996), Vol. 17
Acute sensitivity of first trimester placental tissue to oxygen tension in vitro. Adrian Watson, Marion Palmer, Eric Jauniaux and Graham Burton, Department of Anatomy, University of Cambridge and University College Hospital, London, UK.
When maintaining first trimester human placental villi in organ culture we observed rapid degeneration of the syncytium within 24 hours, despite excellent viability of the cytotrophoblastic and stromal cell types. It was discovered that after handling under ambient conditions mitochondrial activity had all but ceased within the syncytium by 2 hours post-termination. This situation could only be affected by using tissue of 14 weeks-plus gestational age, when the syncjXium showed muih improved survival and mitochondrial activitv. The sueed of the effect on
i I
mitochondria led us to consider that oxidative mechanisms may be the cause of syncytial death. We therefore attempted to maintain villi under low oxygen conditions (2.5%) rather than ambient levels (2 1%). We found that syncytial survival was reproducibly improved and mitochondrial activity retained at time zero levels for 6 hours and more. This effect could only be guaranteed if the tissue was placed into the low oxygen environment within a very short time following removal from the uterus. Furthermore, we observed that neither the copper/zinc nor the manganese forms of superoxide dismutase were present in detectable quantities within the syncytium of 8 and 9 week tissue and only in certain areas of lo-12 week tissue. However, 14-16 week tissue showed considerable amounts of Cu/Zn SOD in the svncvtiotroohoblast. At all sees (8-16 weeks) Cu/Zn SOD ias ietecte‘d in the cytotrophvobl&ts and the ‘stromal cells. These results are in agreement with the proposal that the first trimester of gestation occurs under a low oxygen tension. Supported by the Wellcome Trust (03805 l/Z/93/Z/l S).
CONTROL OF OXYGEN DELIVERY IN DUAL PERFUSION OF AN ISOLATED HUMAN PLACENTAL LOBULE. B. M. Polliotti, AS. Bukhtiar, L. Jessee, C. van den Revdt. D. Comish and R.K. Miller - Deots of Obstetrics and Gyhecology and Environmental Medicine, University of Rochester Medical Center, NY, & *Dept of Pediatrics, Emory University, Atlanta, Georgia, USA.
The efficiency of oxygenation in the human placenta under dual perfusion conditions has always been an important issue in studying normal placental function as well as hypoxia and toxicity. The aim of this study was to investigate improvements in the control of oxygen delivery to the dually perfused isolated human cotyledon using a membrane oxygenator designed for cardiopulmonary bypass applications.
A A&a Silo.+S 0.3 membrane oxygenator (Senko Medical Instruments, Tokyo, Japan) replaces -traditional oxygenation systems (bubbling, rotation, etc.) used to deliver gases to both the maternal and fetal perfusate. Perfusate consists of Ml99 tissue culture medium supplemented with dextran, glucose, L-glutamine, sodium bicarbonate, heparin and antibiotics. Intensive measurements have been done using different concentrations of O2 (0% to 95% 02, with 5% Cd,, balanced with N7). Maternal oerfusate can be 0, “loaded” from 70 to 650
- , ‘.
mmHg in a strictly linear manner by using this membrane oxygenator with different concentrations of 02 in the gas mixture. Without any oxygen carrier (red blood cells, cytochromes or flurocarbons), this membrane oxygenator allows a very precise control of the oxygen provided to the maternal circuit.
Studies requiring hypoxia conditions can be achieved more easily with this device. In addition, this oxygenation system protects the compound (e.g., proteins) added in the perfusate from being degraded or modified by the gases. (Supported in part by NIH Grants - ES 02774 and ES 01247).
The Effects of A4aternal Smoking on Placental Oxygell Transfer. P.G.Bush’, T.M.Mayhew2, D.R.Abramovich3, M.D.Burke’, P.J.Aggett’, & K.R.Page’. Departments of ‘Biomedical Sciences, and 30bstetrics and Gynaecology, Aberdeen University, Aberdeen, 2Human Morphology, Nottingham University, Nottingham, and “Pharmaceutical Sciences, De Montford University, Leicester, ‘Institute of Food Research, Norwich, United Kingdom.
Maternal smoking may have an adverse effect on the transport of respiratory gases to the fetus via changes in placental morphology. In this study, placentas were obtained at delivery from healthy uncomplicated pregnancies and grouped according to maternal smoking habit. Random tissue samples were obtained and following fixation and section staining, structural quantities were estimated and used to calculate the total diffusive conductance for oxygen Dp (Mayhew et al. 1990). Significant morphological changes were observed (including the arithmetic mean of stromal thickness which increased). However, there was no significant change in Dp (cm3 ) 0,. mid’. kPd’) mean (SEM): 17.3 (2.2) (n=ll) in non-smokers and 15.0 (0.8) (n=27) in smokers. Taking into account fetal weight we found also no difference between speicific difisive conductances. Thus despite changes in placental morphology the apparent 13% decline in oxygen diffusive conductance did not attain statistical significance. (Supported by the Tobacco Products Research Trust). Reference: Mayhew TM, Jackson MR, & Haas JD (1990) Placenta 11.493-503.
