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Pro-interleukin-1 beta and mature interleukin-1 beta in human trophoblast
SPO Abstracts
V o l u m e 176, N u m b e r 1, Part 2 A m J O b s t e t Gynecol
$155
539
DOES MATERNAL DIABETES MODULATE EXPRESSION OF THE HUMAN PLACENTAL GLUT1 GLUCOSE TRANSPORTER? K. Gaittw~, N.P. IllsleyL Department of Obstetrics and Gynecology, UMDNJ - New Jersey Medical School, Newark, NJ. OBJECTIVE: To determine whether maternal diabetes affects expression of the GLUT1 glucose transporter in microvillous and basal nmmbranes from h u m a n placenta. STUDY DESIGN: Placental microvillous and basal membranes (MVM, BM) were prepared from 6 normal and 21 diabetic, term pregnancies. Transporter expression was analyzed by slot-blotting of the membranes using a GLUTl-specific antibody and quantified by densitometry. Levels of expression were compared by" ANOVA. RESULTS: Although no difference was found in MVM G L U T i expression between controls and diet-controlled gestational diabetics, the expression in BM was reduced significantly (p < 0.05) to 50 + 10% of comrol (mean _+ sere; n - 9 ) . Preliminary results showed reductions in insulincontrolled gestational diabetics (39 • 15%; n 3) and pregestational diabetics (44%, 48%; n 2), however flm sample size is clearly limited. CONCLUSIONS: GLUT1 transporter expression on the basal membrane is substantially reduced in the diet-controlled group. The (fetalfacing) basal m e m b r a n e normally has a lower complenmnt of transporters than the microvillous m e m b r a n e and further reductions secondary to maternal diabetes may reflect adaptations designed to regulated fetoplacental growth.
541
ADRENOMEDULLIN, A NEW HYPOTENSIVE PEPTIDE IS EXPRESSED IN MATERNAL DECIDUAL CELLS AND FETAL CELLS I N FIRST TRIMESTER OF PREGNANCY. C Macri MD; M. Miller MD; x K Gray PhD, ~ M. Gallagher MD; A. Martinez PhlY, F. Cuttitta Ptd). ~ Departments of Obstetrics and Gynecology, National Naval Medical Center, Uniformed SmMces University of Health Sciences, and Biomarkers and Prevention Research Branch, National Cancer Institute, National Institutes of Health, Befllesda, Maryland. OBJECTIVE: Our purpose was to determine ifAdrenomednllin (AM), a nmlti-functional regulatory" peptide present in second trimester amniotic fluid, was also fonnd in first trimester hmnan pregnancy, and to determine the site of expression of this potent hormone. STUDY DESIGN: A prospective descriptive study design was utilized after approval by the Institutional Review Board. Maternal decidual cells and fetal cells were obtained at the time of chorionic villus sampling (CVS) fi-ona three ongoing pregnancies. Maternal tissue was dissected from the fetal specimen; this tissue is normally discarded to avoid maternal cell contamination of the fetal specimen. Both maternal and ti~tal cells were cultured and evaluated for AM expression using Immunocytoehemistry, Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR), and in situ RT-PCR. RESULTS: Three CVS specimens were analyzed. AM was expressed at high levels by both fire maternal decidual cells and by the fetal chorionic villus ceils. CONCLUSIONS: Adrenomedullin is found in human maternal decidual cells, and in fetal cells in the first trimester of pregnancy. Currently, the function of adrenomedullin in h m n a n pregnancy is not known. Further analysis of AM expression by maternal cells and placental tissue may help to identify the biologic function of this peptide.
540
EXPRESSION OF AN ANGIOGENIC FACTOR, VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN SECOND TRIMESTER A M N I O T I C FLUID AND TERM AMNIOTIC MEMBRANE Charles MacrO, Michael Gallagher, Prabir Chakraboty~, Annette MitcheIP, Karen D. Graf , Departments of OB-GYN, National Naval Medical Center and Uniformed Services University of Health Sciences x, Bethesda, Maryland. OBJECTIVE: To determine if Vascular Endothelial Growth Factor (VEGF), a h u m a n angiogenic factor is present in second trimester amniotic fluid (AF), and ifVEGF and its receptors VEGF 1 (Fh) and VEGF 2 (Flk) are expressed in term human amnion. STUDY DESIGN: A prospective descriptive study of 55 patients undergoing anmiocentesis following genetic counseling, ultrasound, and informed consent was pertormed. VEGF expression in AF was determined using an ELISA assay specific for hmnan VEGF (R&D systems). Fetal membrmles fi-om six term pregnant patients were studied following delivery" for VEGF and its receptors Flt and Flk by immunohistochemistry and RT-PCR. RESULTS: VEGF was detectable in 7 of 55 AF specimens analyzed (11-80 p g / m l ) , and not detectable in 48 (<10 p g / m l ) . Immunocytochenfistry, Western blot analysis and RT-PCR furfller established the expression of VEGF protein and mRNA in fetal amniotic membranes. Protein localization revealed intense VEGF expression in amniotic epkhelimn and in a subpopulation of decidual cells. The VEGF receptor 1 (Fit) was highly expressed in the same cells, but the VEGF receptor 2 (Flk) expression was considerably lower. Variability in the absolute intensity of immunostaining for these proteins between patients may reflect different disease states. PCR analysis revealed transcripts for VEGF and both receptors in all m e m b r a n e sections. CONCLUSIONS: We detected VEGF in 7 of 55 second trtinester AF specimens and showed that the components of the VEGF receptor pathway are expressed in the amniotic epithelium and the decidua. Because VEGF is present in AF and in anmion from term gestations when angiogenesis is not prominent, we believe that VEGF may have other roles in the function of the anmion such as maintenance of vascular permeability and secretion.
542
PRO-INTERLEUKIN-1 BETA AND MATURE INTERLEUKIN-1 BETA IN HUMAN TROPHOBLAST. L M. Vettraino, D. M. Nelson, D.D. Chaplinx, Dept. Ob/Gyn, Washington University, St. Louis, Mo. OBJECTIVE: Interleukin-lbeta (IL-I[3) must be cleaved from the inactive intracellular 31 kD precursor (pro-IL-1 [3) by the protease Interleukin-1 [3 Converting Enzylne (ICE) to the 17kD mature form (mat-IL-113) to have cytokine activity. This investigation evaluates the amounts of pro-IL-1 [3 and mat-IL-l[3 present in h u m a n chorioamnion, decidua and villous trophoblast. STUDY DESIGN: H u m a n placentas were obtained from uncomplicated term vaginal deliveries ( n - 3 ) , scheduled cesarean sections (n=2), preterm vaginal delivery" ( n - 2 ) , and deliveries complicated by chorioamnionitis (n 2). The chorioamnion, decidua, and villous trophoblast were separated from each placenta. The separated tissues were prepared for Western blot analysis. Protein was assayed by the Lowry method, resolved on a 12% SDS-polyacrylamide gel, and transferred to nitrocellulose paper by electroblotting. The nitrocellulose filter was incubated with polyclonal rabbit anti-hmnan IL-l[3 antibody. Blots were developed with the Enhanced Chemiluminescence (ECL) kit. Autoradiography was perfornmd. RESULTS: Signal for pro-IL-1 [3 was seen in all tissue types studied. Signal for lnat-IL-ll3 was seen consistently only in specimens obtained from pregnancies complicated by preterm delivery and choriomnnionitis. Tissues recovered from placental specimens resulting from preterm delivery were associated with the most intense signals for pro-IL-l[3 and mat-IL-l[3 in all blots. The chorioanmion showed greater signal for mat-IL-l[3 than the villous trophoblast frmn the corresponding placental specimen. CONCLUSION: The constitutive expression of pro-IL-l[3 in h u m a n trophoblast indicates that this protein selves a generalized function in the villous. However, pathological processes in human parturition are accompanied by the greatest amounts of the active mat-IL-l[3. Modulators of ICE are critical in the control of these pathologic processes. In addition, the chorioamnion, in closest proximity to the decidua basalis mad myometrium, has an important role in those actions necessitating mat-IL-113. These actions include prostaglandin release known to be important in the initiation of labor.
V o l u m e 176, N u m b e r 1, Part 2 A m J O b s t e t Gynecol
$155
539
DOES MATERNAL DIABETES MODULATE EXPRESSION OF THE HUMAN PLACENTAL GLUT1 GLUCOSE TRANSPORTER? K. Gaittw~, N.P. IllsleyL Department of Obstetrics and Gynecology, UMDNJ - New Jersey Medical School, Newark, NJ. OBJECTIVE: To determine whether maternal diabetes affects expression of the GLUT1 glucose transporter in microvillous and basal nmmbranes from h u m a n placenta. STUDY DESIGN: Placental microvillous and basal membranes (MVM, BM) were prepared from 6 normal and 21 diabetic, term pregnancies. Transporter expression was analyzed by slot-blotting of the membranes using a GLUTl-specific antibody and quantified by densitometry. Levels of expression were compared by" ANOVA. RESULTS: Although no difference was found in MVM G L U T i expression between controls and diet-controlled gestational diabetics, the expression in BM was reduced significantly (p < 0.05) to 50 + 10% of comrol (mean _+ sere; n - 9 ) . Preliminary results showed reductions in insulincontrolled gestational diabetics (39 • 15%; n 3) and pregestational diabetics (44%, 48%; n 2), however flm sample size is clearly limited. CONCLUSIONS: GLUT1 transporter expression on the basal membrane is substantially reduced in the diet-controlled group. The (fetalfacing) basal m e m b r a n e normally has a lower complenmnt of transporters than the microvillous m e m b r a n e and further reductions secondary to maternal diabetes may reflect adaptations designed to regulated fetoplacental growth.
541
ADRENOMEDULLIN, A NEW HYPOTENSIVE PEPTIDE IS EXPRESSED IN MATERNAL DECIDUAL CELLS AND FETAL CELLS I N FIRST TRIMESTER OF PREGNANCY. C Macri MD; M. Miller MD; x K Gray PhD, ~ M. Gallagher MD; A. Martinez PhlY, F. Cuttitta Ptd). ~ Departments of Obstetrics and Gynecology, National Naval Medical Center, Uniformed SmMces University of Health Sciences, and Biomarkers and Prevention Research Branch, National Cancer Institute, National Institutes of Health, Befllesda, Maryland. OBJECTIVE: Our purpose was to determine ifAdrenomednllin (AM), a nmlti-functional regulatory" peptide present in second trimester amniotic fluid, was also fonnd in first trimester hmnan pregnancy, and to determine the site of expression of this potent hormone. STUDY DESIGN: A prospective descriptive study design was utilized after approval by the Institutional Review Board. Maternal decidual cells and fetal cells were obtained at the time of chorionic villus sampling (CVS) fi-ona three ongoing pregnancies. Maternal tissue was dissected from the fetal specimen; this tissue is normally discarded to avoid maternal cell contamination of the fetal specimen. Both maternal and ti~tal cells were cultured and evaluated for AM expression using Immunocytoehemistry, Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR), and in situ RT-PCR. RESULTS: Three CVS specimens were analyzed. AM was expressed at high levels by both fire maternal decidual cells and by the fetal chorionic villus ceils. CONCLUSIONS: Adrenomedullin is found in human maternal decidual cells, and in fetal cells in the first trimester of pregnancy. Currently, the function of adrenomedullin in h m n a n pregnancy is not known. Further analysis of AM expression by maternal cells and placental tissue may help to identify the biologic function of this peptide.
540
EXPRESSION OF AN ANGIOGENIC FACTOR, VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN SECOND TRIMESTER A M N I O T I C FLUID AND TERM AMNIOTIC MEMBRANE Charles MacrO, Michael Gallagher, Prabir Chakraboty~, Annette MitcheIP, Karen D. Graf , Departments of OB-GYN, National Naval Medical Center and Uniformed Services University of Health Sciences x, Bethesda, Maryland. OBJECTIVE: To determine if Vascular Endothelial Growth Factor (VEGF), a h u m a n angiogenic factor is present in second trimester amniotic fluid (AF), and ifVEGF and its receptors VEGF 1 (Fh) and VEGF 2 (Flk) are expressed in term human amnion. STUDY DESIGN: A prospective descriptive study of 55 patients undergoing anmiocentesis following genetic counseling, ultrasound, and informed consent was pertormed. VEGF expression in AF was determined using an ELISA assay specific for hmnan VEGF (R&D systems). Fetal membrmles fi-om six term pregnant patients were studied following delivery" for VEGF and its receptors Flt and Flk by immunohistochemistry and RT-PCR. RESULTS: VEGF was detectable in 7 of 55 AF specimens analyzed (11-80 p g / m l ) , and not detectable in 48 (<10 p g / m l ) . Immunocytochenfistry, Western blot analysis and RT-PCR furfller established the expression of VEGF protein and mRNA in fetal amniotic membranes. Protein localization revealed intense VEGF expression in amniotic epkhelimn and in a subpopulation of decidual cells. The VEGF receptor 1 (Fit) was highly expressed in the same cells, but the VEGF receptor 2 (Flk) expression was considerably lower. Variability in the absolute intensity of immunostaining for these proteins between patients may reflect different disease states. PCR analysis revealed transcripts for VEGF and both receptors in all m e m b r a n e sections. CONCLUSIONS: We detected VEGF in 7 of 55 second trtinester AF specimens and showed that the components of the VEGF receptor pathway are expressed in the amniotic epithelium and the decidua. Because VEGF is present in AF and in anmion from term gestations when angiogenesis is not prominent, we believe that VEGF may have other roles in the function of the anmion such as maintenance of vascular permeability and secretion.
542
PRO-INTERLEUKIN-1 BETA AND MATURE INTERLEUKIN-1 BETA IN HUMAN TROPHOBLAST. L M. Vettraino, D. M. Nelson, D.D. Chaplinx, Dept. Ob/Gyn, Washington University, St. Louis, Mo. OBJECTIVE: Interleukin-lbeta (IL-I[3) must be cleaved from the inactive intracellular 31 kD precursor (pro-IL-1 [3) by the protease Interleukin-1 [3 Converting Enzylne (ICE) to the 17kD mature form (mat-IL-113) to have cytokine activity. This investigation evaluates the amounts of pro-IL-1 [3 and mat-IL-l[3 present in h u m a n chorioamnion, decidua and villous trophoblast. STUDY DESIGN: H u m a n placentas were obtained from uncomplicated term vaginal deliveries ( n - 3 ) , scheduled cesarean sections (n=2), preterm vaginal delivery" ( n - 2 ) , and deliveries complicated by chorioamnionitis (n 2). The chorioamnion, decidua, and villous trophoblast were separated from each placenta. The separated tissues were prepared for Western blot analysis. Protein was assayed by the Lowry method, resolved on a 12% SDS-polyacrylamide gel, and transferred to nitrocellulose paper by electroblotting. The nitrocellulose filter was incubated with polyclonal rabbit anti-hmnan IL-l[3 antibody. Blots were developed with the Enhanced Chemiluminescence (ECL) kit. Autoradiography was perfornmd. RESULTS: Signal for pro-IL-1 [3 was seen in all tissue types studied. Signal for lnat-IL-ll3 was seen consistently only in specimens obtained from pregnancies complicated by preterm delivery and choriomnnionitis. Tissues recovered from placental specimens resulting from preterm delivery were associated with the most intense signals for pro-IL-l[3 and mat-IL-l[3 in all blots. The chorioanmion showed greater signal for mat-IL-l[3 than the villous trophoblast frmn the corresponding placental specimen. CONCLUSION: The constitutive expression of pro-IL-l[3 in h u m a n trophoblast indicates that this protein selves a generalized function in the villous. However, pathological processes in human parturition are accompanied by the greatest amounts of the active mat-IL-l[3. Modulators of ICE are critical in the control of these pathologic processes. In addition, the chorioamnion, in closest proximity to the decidua basalis mad myometrium, has an important role in those actions necessitating mat-IL-113. These actions include prostaglandin release known to be important in the initiation of labor.