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Effect of induced inflammation in mice during pregnancy
Abstracts / Placenta 34 (2013) A1–A99
P1.61. EFFECT OF INDUCED INFLAMMATION IN MICE DURING PREGNANCY Bruno Zavan 1, Eliana M. Almeida 1, Eliana M.O. Lippe 2, Alexandre Giusti-Paiva 1, Aureo T. Yamada 2, Valdemar A. Paffaro-Jr 1 1
Federal University of Alfenas, Alfenas, MG, Brazil; 2 University of Campinas, Campinas, SP, Brazil Objectives: To evaluate the LPS effect on pregnant mouse behavior, nitric oxide (NO) levels and uterine morphology. Methods: Under Ethics Committee approval, mice on gestation day (gd) 10 received intraperitoneally LPS (0.5-1mg/animal) and were euthanized after 0.5h, 1h, 2h and 4h. Pretreatment (0.5h before LPS) with L-NAME (2mg/ mL) were also performed in an additional group which was euthanized similarly to the LPS group. All animals were evaluated for behavioral, uterine natural killer cell (uNK) quantification and NO colorimetric studies. Two groups of pregnant mice (dg10) were added, which received respectively only Nimesulide (5mg/kg) and Nimesulide 30min before LPS. Histological sections of the embryo implantation sites (collected 2h after treatment) were subjected to DBA lectin cytochemestry. Results: Feverish and anxiety-like behavior were observed 2h after LPS. Apathy, exploratory capacity reduction and depressive-like behavior were noted 1h after LPS. LPS causes alterations in the uNK DBA lectin staining. A significant augment of immature uNK was seen in myometrium concomitantly to decreasing in mature and senescent uNK cells number while LPS-altered uNK increases. Sickness behavior, uNK number and NO serum levels were higher at 2h of LPS, but not as high as seen in L-NAME group. The uNK cells from Nimesulide-treated mice showed less altereduNK compared to LPS-treated mice. Conclusions: The present work demonstrates the damaging effects of induced inflammation during pregnancy, with strong evidence of prostaglandin participation in this process. These results also suggest that the NO levels can be indicative of sickness behavior and uterine morphophysiological changes in LPS-treated pregnant mice. http://dx.doi.org/10.1016/j.placenta.2013.06.098
P1.62. FUNCTIONAL TOLL-LIKE RECEPTORS IN PRIMARY FIRST TRIMESTER TROPHOBLASTS Line Haugstad Tangerås 1, 2, Guro Sannerud Stødle 1, 2, Guro Dalheim Olsen 1, Ann-Helen Leknes 1, Astrid Gundersen 1, Bente Skei 1, Anne Jorunn Vikdal 1, Liv Ryan 1, Bjørg Steinkjer 1, Merete F. Myklebost 3, Mette Langaas 4, Rigmor Austgulen 1, Ann-Charlotte Iversen 1 1
Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; 2 Liaison Committee between the Central Norway Regional Health Authority (RHA) and NTNU, Trondheim, Norway; 3 Department of Gynaecology and Obstetrics, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway; 4 Department of Mathematical Sciences, NTNU, Trondheim, Norway Objectives: In a successful pregnancy, the first trimester is characterised by a mild pro-inflammatory environment. However, excessive inflammation threatens placental development and function, potentially resulting in adverse pregnancy outcomes such as preeclampsia. The human family of ten Toll-like receptors (TLRs) is crucial for initiating inflammation by directly sensing noxious stimuli from infections and tissue damage, playing a central role in inflammatory diseases. Recent findings of TLRs in trophoblasts imply relevance to pregnancy complications, but the knowledge is limited, particularly for early gestation. The aim of this study was to examine gene expression and function of all ten human TLRs in primary trophoblasts from first trimester placentas.
A33
Methods: Primary trophoblasts were isolated from first trimester placentas (n ¼ 6) by enzyme degradation and density gradient centrifugation. The first trimester trophoblast cell line BeWo was included for comparison. Gene expression of TLR1-10 was quantified by real-time quantitative reverse-transcription polymerase chain reaction in primary trophoblasts and BeWo cells. Trophoblasts were stimulated with ligands for TLR1-9 and production of pro-inflammatory cytokines analysed by multiplex immunoassay. Results: Primary first trimester trophoblasts showed gene expression of all ten TLRs, with varying TLR gene expression profiles between individual trophoblast populations. Ligand-induced activation of TLR2/1, TLR3, TLR4, TLR5 and TLR9 increased production of the pro-inflammatory cytokines interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF) and/or interferon-g inducible protein 10 (IP-10), in primary first trimester trophoblasts. Compared to BeWo cells, the primary trophoblasts expressed higher levels of all ten TLR mRNAs and responded more potently to TLR ligand-induced activation. Conclusion: The broad expression of functional TLRs in primary first trimester trophoblasts suggests an important inflammatory role for early gestation trophoblasts. http://dx.doi.org/10.1016/j.placenta.2013.06.099
P1.63. MECHANISMS IN ENDOVASCULAR DIFFERENTIATION TROPHOBLAST AND ITS REGULATION BY DECORIN
OF
THE
Peeyush Lala, Gannareddy Girish University of Western Ontario, London, Ontario, N6A5C1, Canada Introduction: Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and adopt an endothelial phenotype to modify utero-placental arteries. These events are compromised in pre-eclampsia, a trophoblast hypo-invasive disease in pregnancy. EVT invasion is stringently regulated in-situ by many locally-produced factors. We found that Decorin (DCN), a leucine rich proteoglycan produced by the decidua, negatively regulates EVT functions by binding to EGF-R, IGF-R1 and VEGFR-2. VEGFR-2 binding site in DCN was localized to its leucine-richrepeat (LLR)-5 domain, antagonising VEGF actions. However, mechanisms underlying endovascular differentiation of the EVT and its possible regulation by DCN remain unclear. Objectives and approaches: Present study utilized the first trimester human EVT cell line, HTR-8/SVneo (1) To test whether VEGF promoted endovascular differentiation of the EVT, and if so, (2) To identify molecular mechanisms underlying decorin antagonism of VEGF–mediated EVT cell migration and endovascular differentiation. The latter was measured by the ability of the EVT (a) to form endothelial-like tubes on matrigel and (b) to express endothelial activation markers VE-cadherin and b-catenin. Results: (1) Exogenous VEGF stimulated migration, endothelial-like tube formation and upregulated VE-cadherin and b-catenin in EVT cells. This upregulation was VEGFR-2 dependent, shown by siRNA-mediated knock down of VEGFR-2 in the EVT. (2) DCN blocked all of the above VEGFstimulated events. (3) Exogenous VEGF stimulated phosphorylation of MAPKs (P38,ERK1/2) in EVT cells, and the stimulation was blocked in both cases by decorin. Both pathways contributed independently to VEGFinduced EVT migration and tube formation. VEGF-mediated upregulation of VE-cadherin and b-catenin in the EVT cells were blocked by both MAPK inhibitors, indicating MAPK dependence. Conclusion: Decorin antagonizes VEGF-stimulation of trophoblast migration and endovascular differentiation by interfering with p38MAPK and ERK1/2 activation. Significance: we are exploring whether decorin over-activity underlies pathogenesis of pre-eclampsia in which endovascular differentiation of the EVT is compromised. (Supported by a CIHR grant to PKL) http://dx.doi.org/10.1016/j.placenta.2013.06.100
P1.61. EFFECT OF INDUCED INFLAMMATION IN MICE DURING PREGNANCY Bruno Zavan 1, Eliana M. Almeida 1, Eliana M.O. Lippe 2, Alexandre Giusti-Paiva 1, Aureo T. Yamada 2, Valdemar A. Paffaro-Jr 1 1
Federal University of Alfenas, Alfenas, MG, Brazil; 2 University of Campinas, Campinas, SP, Brazil Objectives: To evaluate the LPS effect on pregnant mouse behavior, nitric oxide (NO) levels and uterine morphology. Methods: Under Ethics Committee approval, mice on gestation day (gd) 10 received intraperitoneally LPS (0.5-1mg/animal) and were euthanized after 0.5h, 1h, 2h and 4h. Pretreatment (0.5h before LPS) with L-NAME (2mg/ mL) were also performed in an additional group which was euthanized similarly to the LPS group. All animals were evaluated for behavioral, uterine natural killer cell (uNK) quantification and NO colorimetric studies. Two groups of pregnant mice (dg10) were added, which received respectively only Nimesulide (5mg/kg) and Nimesulide 30min before LPS. Histological sections of the embryo implantation sites (collected 2h after treatment) were subjected to DBA lectin cytochemestry. Results: Feverish and anxiety-like behavior were observed 2h after LPS. Apathy, exploratory capacity reduction and depressive-like behavior were noted 1h after LPS. LPS causes alterations in the uNK DBA lectin staining. A significant augment of immature uNK was seen in myometrium concomitantly to decreasing in mature and senescent uNK cells number while LPS-altered uNK increases. Sickness behavior, uNK number and NO serum levels were higher at 2h of LPS, but not as high as seen in L-NAME group. The uNK cells from Nimesulide-treated mice showed less altereduNK compared to LPS-treated mice. Conclusions: The present work demonstrates the damaging effects of induced inflammation during pregnancy, with strong evidence of prostaglandin participation in this process. These results also suggest that the NO levels can be indicative of sickness behavior and uterine morphophysiological changes in LPS-treated pregnant mice. http://dx.doi.org/10.1016/j.placenta.2013.06.098
P1.62. FUNCTIONAL TOLL-LIKE RECEPTORS IN PRIMARY FIRST TRIMESTER TROPHOBLASTS Line Haugstad Tangerås 1, 2, Guro Sannerud Stødle 1, 2, Guro Dalheim Olsen 1, Ann-Helen Leknes 1, Astrid Gundersen 1, Bente Skei 1, Anne Jorunn Vikdal 1, Liv Ryan 1, Bjørg Steinkjer 1, Merete F. Myklebost 3, Mette Langaas 4, Rigmor Austgulen 1, Ann-Charlotte Iversen 1 1
Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway; 2 Liaison Committee between the Central Norway Regional Health Authority (RHA) and NTNU, Trondheim, Norway; 3 Department of Gynaecology and Obstetrics, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway; 4 Department of Mathematical Sciences, NTNU, Trondheim, Norway Objectives: In a successful pregnancy, the first trimester is characterised by a mild pro-inflammatory environment. However, excessive inflammation threatens placental development and function, potentially resulting in adverse pregnancy outcomes such as preeclampsia. The human family of ten Toll-like receptors (TLRs) is crucial for initiating inflammation by directly sensing noxious stimuli from infections and tissue damage, playing a central role in inflammatory diseases. Recent findings of TLRs in trophoblasts imply relevance to pregnancy complications, but the knowledge is limited, particularly for early gestation. The aim of this study was to examine gene expression and function of all ten human TLRs in primary trophoblasts from first trimester placentas.
A33
Methods: Primary trophoblasts were isolated from first trimester placentas (n ¼ 6) by enzyme degradation and density gradient centrifugation. The first trimester trophoblast cell line BeWo was included for comparison. Gene expression of TLR1-10 was quantified by real-time quantitative reverse-transcription polymerase chain reaction in primary trophoblasts and BeWo cells. Trophoblasts were stimulated with ligands for TLR1-9 and production of pro-inflammatory cytokines analysed by multiplex immunoassay. Results: Primary first trimester trophoblasts showed gene expression of all ten TLRs, with varying TLR gene expression profiles between individual trophoblast populations. Ligand-induced activation of TLR2/1, TLR3, TLR4, TLR5 and TLR9 increased production of the pro-inflammatory cytokines interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF) and/or interferon-g inducible protein 10 (IP-10), in primary first trimester trophoblasts. Compared to BeWo cells, the primary trophoblasts expressed higher levels of all ten TLR mRNAs and responded more potently to TLR ligand-induced activation. Conclusion: The broad expression of functional TLRs in primary first trimester trophoblasts suggests an important inflammatory role for early gestation trophoblasts. http://dx.doi.org/10.1016/j.placenta.2013.06.099
P1.63. MECHANISMS IN ENDOVASCULAR DIFFERENTIATION TROPHOBLAST AND ITS REGULATION BY DECORIN
OF
THE
Peeyush Lala, Gannareddy Girish University of Western Ontario, London, Ontario, N6A5C1, Canada Introduction: Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and adopt an endothelial phenotype to modify utero-placental arteries. These events are compromised in pre-eclampsia, a trophoblast hypo-invasive disease in pregnancy. EVT invasion is stringently regulated in-situ by many locally-produced factors. We found that Decorin (DCN), a leucine rich proteoglycan produced by the decidua, negatively regulates EVT functions by binding to EGF-R, IGF-R1 and VEGFR-2. VEGFR-2 binding site in DCN was localized to its leucine-richrepeat (LLR)-5 domain, antagonising VEGF actions. However, mechanisms underlying endovascular differentiation of the EVT and its possible regulation by DCN remain unclear. Objectives and approaches: Present study utilized the first trimester human EVT cell line, HTR-8/SVneo (1) To test whether VEGF promoted endovascular differentiation of the EVT, and if so, (2) To identify molecular mechanisms underlying decorin antagonism of VEGF–mediated EVT cell migration and endovascular differentiation. The latter was measured by the ability of the EVT (a) to form endothelial-like tubes on matrigel and (b) to express endothelial activation markers VE-cadherin and b-catenin. Results: (1) Exogenous VEGF stimulated migration, endothelial-like tube formation and upregulated VE-cadherin and b-catenin in EVT cells. This upregulation was VEGFR-2 dependent, shown by siRNA-mediated knock down of VEGFR-2 in the EVT. (2) DCN blocked all of the above VEGFstimulated events. (3) Exogenous VEGF stimulated phosphorylation of MAPKs (P38,ERK1/2) in EVT cells, and the stimulation was blocked in both cases by decorin. Both pathways contributed independently to VEGFinduced EVT migration and tube formation. VEGF-mediated upregulation of VE-cadherin and b-catenin in the EVT cells were blocked by both MAPK inhibitors, indicating MAPK dependence. Conclusion: Decorin antagonizes VEGF-stimulation of trophoblast migration and endovascular differentiation by interfering with p38MAPK and ERK1/2 activation. Significance: we are exploring whether decorin over-activity underlies pathogenesis of pre-eclampsia in which endovascular differentiation of the EVT is compromised. (Supported by a CIHR grant to PKL) http://dx.doi.org/10.1016/j.placenta.2013.06.100