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PP028. Serum cytokine profile in relation to the clinical features and laboratory parameters in women with preeclampsia
Poster Presentations / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 3 (2013) 67–99
Objectives: To determine circulating levels of cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia, and to investigate their relationship to the clinical features and laboratory parameters of the patients. Methods: Serum levels of IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, IFN-gamma, TNFalpha, TGF-beta1, IP-10, MCP-1, ICAM-1 and VCAM-1 were measured in 60 preeclamptic, 60 healthy pregnant and 59 healthy non-pregnant women. Results: In addition to a shift towards Th1-type immunity (increased IL-2/IL-4 and IFN-gamma/IL-4 ratios), levels of the pro-inflammatory cytokines IL-6 and TNF-alpha, the chemokines IL-8, IP-10 and MCP-1, as well as the adhesion molecules ICAM-1 and VCAM-1, were raised in preeclampsia, resulting in an overall pro-inflammatory systemic environment. Increased IP-10, MCP-1, ICAM-1 and VCAM-1 concentrations of preeclamptic patients showed significant correlations with blood pressure values, renal and liver function parameters, as well as with CRP, malondialdehyde, von Willebrand factor antigen and fibronectin levels. Conclusion: Elevated amounts of pro-inflammatory cytokines, chemokines and adhesion molecules in the maternal circulation might play a central role in the excessive systemic inflammatory response, as well as in the generalized endothelial dysfunction characteristics of preeclampsia. doi:10.1016/j.preghy.2013.04.055
PP029. Genetic change of adipose tissue related to inflammation in preeclampsia: Findings under novel culture method Naruse Katsuhiko, Tsunemi Taihei, Onogi Akira, Koike Natsuki, Akasaka Juria, Oi Hidekazu, Kobayashi Hiroshi Introduction: Recently, adipose tissue is suggested to contribute to the inflammatory action in preeclampsia(PE), from peripheral increase of cytokines. However, the mechanism of the action in adipose tissue was not clarified yet. Objectives: In this study, we performed ‘‘Gel bottom-captured’’ adipose tissue culture with PE to show that the adipose tissue is the inflammatory focus in the pathophysiology of PE. Methods: Under informed consent of the patients, omentum from probe laparotomy for ovarian cancer was captured in Peptide HydrogelÒ. After 24 h starvation, tissues were cultured with medium containing 10% of severe PE and healthy pregnant maternal serum (n = 5 each). M30 (Apoptosis) and M65 (all cell death) were measured with ELISA. After mRNA extraction from tissue, quantitative PCR array (Qiagen, Inc.) was performed on all samples. Results: No significant histological differences were found between each culture. However, M30/M65 ratio was increased in PE (p = 0.053). In PCR array, under 2-fold decrease in OSM (p = 0.019) was found, and over 2-fold increase in TLR (p < 0.01) and other inflammatory genes were defined in PE. Conclusion: Inflammatory action in PE via TLR pathway was defined by adipose tissue culture. Apoptosis were
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shown in PE condition, but total tissue injury include necrosis were suggested to be stronger in normal pregnancy. Increase of inflammatory gene suggested that adipose tissue is one of a main organ of systemic inflammation in PE. doi:10.1016/j.preghy.2013.04.056
PP030. Transmission electron microscopy reveals leakage of laeverin into the villous capillaries and ectopic expression in the cytoplasm instead of cell membrane of the trophoblasts in preeclamptic placentas Nystad Mona, Sitras Vasilis, Acharya Ganesh Introduction: Laeverin, a membrane-bound aminopeptidase is specifically expressed in human placenta. We previously reported that mRNA levels of laeverin-gene are upregulated in the placentas of severely preeclamptic women compared to healthy controls. Furthermore, immunofluorescence studies indicated that laeverin is expressed in the cytoplasm rather than the cell membrane of preeclamptic placentas. Objective: To study differences in size and integrity of laeverin protein expressed in preeclamptic and normal placentas, and to investigate sub-cellular localization of laeverin, in the trophoblasts. Methods: Proteins from placental tissue of three severely preeclamptic women and three healthy controls were extracted using Magnabeads in MagNaLyser in T-PER solution. Western blot analysis was done by SDS–polyacrylamide gel electrophoresis and electro-blotting on PVDP membranes. Membranes were developed by Tropix CDPStar and immuno-reactive bands were visualised. Immunoelectronmicroscopy was performed on high pressure freezed (Tokyasu method) tissue samples of three placentas (from1 healthy and 2 severely preeclamptic women). Ultrathin sections were fixed and labeled with primary antibody raised against laeverin, followed by antibody conjugated with 5 nm gold particles (PAG5). Experiments were performed in triplicates and images were taken using a JEM-1010 transmission electron microscope at 50,000 and 70,000 magnifications. Results: Western blot analysis detected laeverin-protein of normal size (approximately 100 kDa) both in normal and preeclamptic placentas. However, laeverin was overexpressed in preeclamptic placentas compared to healthy controls. Immuno-electronmicroscopy revealed presence of laeverin within the capillaries in preeclamptic placentas which was not seen in healthy controls. At a sub-cellular level laeverin was localized on the cell membrane of trophoblasts in healthy placentas. However, in preeclamptic placentas, laeverin was localized in the cytoplasm and in particular in the Golgi apparatus. Conclusions: In preeclamptic placentas, laeverin is overexpressed and it appears to leak into the villous capillaries and localize in the cytoplasm instead of cell membrane of the trophoblasts. doi:10.1016/j.preghy.2013.04.057
Objectives: To determine circulating levels of cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia, and to investigate their relationship to the clinical features and laboratory parameters of the patients. Methods: Serum levels of IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, IFN-gamma, TNFalpha, TGF-beta1, IP-10, MCP-1, ICAM-1 and VCAM-1 were measured in 60 preeclamptic, 60 healthy pregnant and 59 healthy non-pregnant women. Results: In addition to a shift towards Th1-type immunity (increased IL-2/IL-4 and IFN-gamma/IL-4 ratios), levels of the pro-inflammatory cytokines IL-6 and TNF-alpha, the chemokines IL-8, IP-10 and MCP-1, as well as the adhesion molecules ICAM-1 and VCAM-1, were raised in preeclampsia, resulting in an overall pro-inflammatory systemic environment. Increased IP-10, MCP-1, ICAM-1 and VCAM-1 concentrations of preeclamptic patients showed significant correlations with blood pressure values, renal and liver function parameters, as well as with CRP, malondialdehyde, von Willebrand factor antigen and fibronectin levels. Conclusion: Elevated amounts of pro-inflammatory cytokines, chemokines and adhesion molecules in the maternal circulation might play a central role in the excessive systemic inflammatory response, as well as in the generalized endothelial dysfunction characteristics of preeclampsia. doi:10.1016/j.preghy.2013.04.055
PP029. Genetic change of adipose tissue related to inflammation in preeclampsia: Findings under novel culture method Naruse Katsuhiko, Tsunemi Taihei, Onogi Akira, Koike Natsuki, Akasaka Juria, Oi Hidekazu, Kobayashi Hiroshi Introduction: Recently, adipose tissue is suggested to contribute to the inflammatory action in preeclampsia(PE), from peripheral increase of cytokines. However, the mechanism of the action in adipose tissue was not clarified yet. Objectives: In this study, we performed ‘‘Gel bottom-captured’’ adipose tissue culture with PE to show that the adipose tissue is the inflammatory focus in the pathophysiology of PE. Methods: Under informed consent of the patients, omentum from probe laparotomy for ovarian cancer was captured in Peptide HydrogelÒ. After 24 h starvation, tissues were cultured with medium containing 10% of severe PE and healthy pregnant maternal serum (n = 5 each). M30 (Apoptosis) and M65 (all cell death) were measured with ELISA. After mRNA extraction from tissue, quantitative PCR array (Qiagen, Inc.) was performed on all samples. Results: No significant histological differences were found between each culture. However, M30/M65 ratio was increased in PE (p = 0.053). In PCR array, under 2-fold decrease in OSM (p = 0.019) was found, and over 2-fold increase in TLR (p < 0.01) and other inflammatory genes were defined in PE. Conclusion: Inflammatory action in PE via TLR pathway was defined by adipose tissue culture. Apoptosis were
77
shown in PE condition, but total tissue injury include necrosis were suggested to be stronger in normal pregnancy. Increase of inflammatory gene suggested that adipose tissue is one of a main organ of systemic inflammation in PE. doi:10.1016/j.preghy.2013.04.056
PP030. Transmission electron microscopy reveals leakage of laeverin into the villous capillaries and ectopic expression in the cytoplasm instead of cell membrane of the trophoblasts in preeclamptic placentas Nystad Mona, Sitras Vasilis, Acharya Ganesh Introduction: Laeverin, a membrane-bound aminopeptidase is specifically expressed in human placenta. We previously reported that mRNA levels of laeverin-gene are upregulated in the placentas of severely preeclamptic women compared to healthy controls. Furthermore, immunofluorescence studies indicated that laeverin is expressed in the cytoplasm rather than the cell membrane of preeclamptic placentas. Objective: To study differences in size and integrity of laeverin protein expressed in preeclamptic and normal placentas, and to investigate sub-cellular localization of laeverin, in the trophoblasts. Methods: Proteins from placental tissue of three severely preeclamptic women and three healthy controls were extracted using Magnabeads in MagNaLyser in T-PER solution. Western blot analysis was done by SDS–polyacrylamide gel electrophoresis and electro-blotting on PVDP membranes. Membranes were developed by Tropix CDPStar and immuno-reactive bands were visualised. Immunoelectronmicroscopy was performed on high pressure freezed (Tokyasu method) tissue samples of three placentas (from1 healthy and 2 severely preeclamptic women). Ultrathin sections were fixed and labeled with primary antibody raised against laeverin, followed by antibody conjugated with 5 nm gold particles (PAG5). Experiments were performed in triplicates and images were taken using a JEM-1010 transmission electron microscope at 50,000 and 70,000 magnifications. Results: Western blot analysis detected laeverin-protein of normal size (approximately 100 kDa) both in normal and preeclamptic placentas. However, laeverin was overexpressed in preeclamptic placentas compared to healthy controls. Immuno-electronmicroscopy revealed presence of laeverin within the capillaries in preeclamptic placentas which was not seen in healthy controls. At a sub-cellular level laeverin was localized on the cell membrane of trophoblasts in healthy placentas. However, in preeclamptic placentas, laeverin was localized in the cytoplasm and in particular in the Golgi apparatus. Conclusions: In preeclamptic placentas, laeverin is overexpressed and it appears to leak into the villous capillaries and localize in the cytoplasm instead of cell membrane of the trophoblasts. doi:10.1016/j.preghy.2013.04.057