RESUMO
Pregnancy-associated plasma protein-A 2 (PAPPA2) is a placental-enriched gene that is important for normal human placentation and defects in the gene can cause complications in pregnancy. Yet the exact expression pattern and role of PAPPA2 in the human fetomaternal interface are not clear. In this study, in situ hybridization (ISH) and immunohistochemistry (IHC) were employed to examine the spatial and temporal expression of PAPPA2 in the human fetomaternal interface. IHC results exhibited wide expression of PAPPA2 in the fetomaternal interface, with placental syncytiatrophoblast (STB) and extravillous trophoblast (EVT) showing strong expression and the cytotrophoblast (CTB) showing weak expression of PAPPA2. These results were confirmed by ISH. Quantitative reverse transcription-polymerase chain reaction and western blot showed the elevation of PAPPA2 in first trimester EVT differentiation and term CTB spontaneous syncytialization. PAPPA2-siRNA transfection significantly depressed the invasion and migration ability of a trophoblast cell line (HTR8/SVneo) in a transwell migration and Matrigel invasion model compared to a negative control siRNA (P < 0.05), also revealing that matrix metalloproteinase 9 (MMP9) secretion is downregulated. This was confirmed using a human first trimester placental villi explant culture model. Our results reveal the spatial and temporal expression of PAPPA2 in the human fetomaternal interface and show the positive regulatory role of PAPPA2 in human trophoblast invasion and migration through the secretion of MMP9.
Assuntos
Movimento Celular/fisiologia , Proteína Plasmática A Associada à Gravidez/biossíntese , Trofoblastos/enzimologia , Linhagem Celular , Células Cultivadas , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/fisiologia , Feminino , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Placentação/fisiologia , Gravidez , Proteína Plasmática A Associada à Gravidez/genética , RNA Mensageiro/genética , Trofoblastos/fisiologiaRESUMO
Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier, however studies about the physiopathology of this process are scarce. Different signal transduction pathways are involved during cell invasion of the parasite. However, the possible role of those processes during tissue infection has not been studied. In the present study we analyzed the modulation of two signal transduction pathways, PLC-γ and ERK1/2 MAPK, during ex vivo infection of human placental chorionic villi explants. Chorionic villi from healthy woman placentas were incubated in the presence or absence of 10(5) or 10(6)T. cruzi trypomastigotes (DM28c strain) with or without specific inhibitors for each pathway. Effective infection was tested determining parasite DNA by PCR. The activation of PLC-γ and ERK1/2 MAPK signaling pathways was determined by western blotting and immunofluorescence. The low concentration of T. cruzi trypomastigotes activates both signaling pathways; however, the high concentration of parasite induces a modest activation of the PLC-γ pathway and impairs the ERK1/2 MAPK pathway activation. Interestingly, inhibition of any of those signaling pathways did not prevent parasite infection, as it was previously shown in cell cultures. We conclude that both signal transduction pathways are modulated during ex vivo T. cruzi infection of human placental chorionic villi explants.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Fosfolipase C gama/metabolismo , Placenta/enzimologia , Placenta/parasitologia , Animais , Chlorocebus aethiops , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/parasitologia , Feminino , Humanos , Gravidez , Transdução de Sinais/fisiologia , Células VeroRESUMO
BACKGROUND: Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier. In the present study we analyzed the participation of matrix metalloproteases (MMPs) in the extracellular matrix (ECM) remodeling during T. cruzi ex vivo infection of human placental chorionic villi explants. METHODS: Chorionic villi from healthy woman placentas were incubated in the presence or absence of 105 or 106 T. cruzi trypomastigotes (Y strain) with or without the MMPs inhibitor doxycycline. Effective infection was tested measuring parasite DNA by real time PCR (qPCR). MMP-2 and MMP-9 expression were determined by western blotting and immunohistochemistry and their activities were measured by zymography. The effect of MMPs on ECM structure was analyzed histochemically. RESULTS: T. cruzi induces the expression and activity of MMP-2 and MMP-9 in chorionic villi. Inhibition of the MMPs prevents the tissue damage induced by T. cruzi and partially decreases the ex vivo infection of the chorionic villi. CONCLUSION: MMPs are partially responsible for the ECM changes observed in human chorionic villi during T. cruzi infection and participate in tissue invasion. On the other hand, MMPs may be part of a local placental antiparasitic mechanism.
Assuntos
Doença de Chagas/imunologia , Vilosidades Coriônicas/enzimologia , Resistência à Doença , Indução Enzimática , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Trypanosoma cruzi/imunologia , Western Blotting , Doença de Chagas/patologia , Doença de Chagas/prevenção & controle , Doença de Chagas/transmissão , Vilosidades Coriônicas/imunologia , Vilosidades Coriônicas/parasitologia , Vilosidades Coriônicas/patologia , DNA de Protozoário/metabolismo , Doxiciclina/farmacologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/parasitologia , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/patogenicidadeRESUMO
It is known that human syncytiotrophoblast (hSCT) actively transports more than 80% of the Ca2+ that goes from maternal to fetal circulation. Transepithelial transport of Ca2+ is carried out through channels, transporters and exchangers located in both microvillous (MVM) and basal (BM) plasma membranes. The plasma membrane Ca-ATPase (PMCA) is the most important mechanism of Ca2+ homeostasis control in the human placenta. In this work, we reexamined the distribution of PMCA in isolated hSCT of term placenta. The PMCA activity was determined in isolated hSCT plasma membranes. A partial characterization of the PMCA activity was performed, including an evaluation of the sensitivity of this enzyme to an in vitro induced lipid peroxidation. Expression of the PMCA in hSCT plasma membranes and tissue sections was investigated using Western blots and immunohistochemistry, respectively. Our study demonstrates, for the first time, a correlation between the activity and structural distribution of PMCA in both MVM and BM of hSCT. It also demonstrates a higher PMCA activity and expression in MVM as compared to BM. Finally, PMCA4 seems to be preferentially distributed in both hSCT plasma membranes, while PMCA1 is shown to be present in the hSCT homogenate. However, the membrane fractions did not show any PMCA1 labeling. Our results must be taken into account in order to propose a new model for the transport of calcium across the hSCT.
Assuntos
Membrana Celular/metabolismo , Vilosidades Coriônicas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Nascimento a Termo/metabolismo , Trofoblastos/metabolismo , Transporte Biológico/fisiologia , Cálcio/metabolismo , Separação Celular , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/ultraestrutura , Feminino , Humanos , Isoenzimas/metabolismo , Microvilosidades/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/fisiologia , Gravidez , Distribuição Tecidual , Extratos de Tecidos/química , Extratos de Tecidos/metabolismo , Trofoblastos/enzimologia , Trofoblastos/ultraestruturaRESUMO
Previous work has demonstrated that PLAP activity decreases in serum and placental villi from term chagasic and diabetic pregnant women. In vitro, T. cruzi induces changes in human syncytiotrophoblast's PLAP. Our aim was to determine if infection with T. cruzi induces changes in PLAP activity in diabetic and chagasic women's placenta, in order to elucidate if PLAP plays a role in the mechanisms of interaction between placenta and T. cruzi, and whether hyperglycemic conditions could worsen the placental infection. Using zymogrammes, Western blot, biochemical and immunohistological techniques, PLAP activity was determined in placental villi from diabetic and chagasic women, and in normal placentas cultured under hyperglycemic conditions with or without trypomastigotes. A significant reduction of PLAP expression was immunologically detected in infected diabetic and normal placental villi cultured under hyperglycemic conditions of 71 and 81%, respectively, compared with controls. A significant decrease of PLAP specific activity was registered in homogenates and in the culture media from both infected diabetic and normal placentas under hyperglycemic conditions (of about 50-70%), and in chagasic ones (of about 87%), when compared with controls. Thus, PLAP might be involved in parasite invasion and diabetic and hyperglycemic placentas could be more susceptible to T. cruzi infection.
Assuntos
Doença de Chagas/enzimologia , Vilosidades Coriônicas/enzimologia , Isoenzimas/metabolismo , Complicações Parasitárias na Gravidez/enzimologia , Gravidez em Diabéticas/enzimologia , Trypanosoma cruzi/isolamento & purificação , Adulto , Fosfatase Alcalina , Animais , Western Blotting , Doença de Chagas/complicações , Vilosidades Coriônicas/parasitologia , Vilosidades Coriônicas/patologia , Técnicas de Cocultura , Feminino , Proteínas Ligadas por GPI , Humanos , Técnicas Imunoenzimáticas , Técnicas de Cultura de Órgãos , Gravidez , Gravidez em Diabéticas/parasitologia , Trypanosoma cruzi/fisiologiaRESUMO
Trypanosoma cruzi induces changes in the protein pattern of human placenta syncytiotrophoblast. Placental alkaline phosphatase (PLAP) is a glycoenzyme anchored to the membrane by a glycosyl-phosphatidylinositol molecule. PLAP activity and its presence was altered by the parasite in cultures of human placental villi and HEp2 cells with T.cruzi. The cells treated before the cultures with agents which affect PILAP or glycosyl-phosphatidylinositol (antibodies, PL-C, genistein, lithium) presented less parasitic invasion than the control ones. It was also observed a modification in the pattern of actine filaments of the host cells infected. We concluded that PLAP would participate in the process of T. cruzi invasion into placental syncitiotrophoblast cells, by a mechanism that involves hydrolysis of the glycosyl-phosphatidylinositol molecules, the activation of tyrosine kinase proteins, the increase of cytosolic calcium and the rearrangement of actine filaments of the host cells.
Assuntos
Fosfatase Alcalina/metabolismo , Doença de Chagas/enzimologia , Placenta/enzimologia , Trypanosoma cruzi/fisiologia , Fosfatase Alcalina/análise , Análise de Variância , Animais , Biomarcadores , Técnicas de Cultura de Células , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/parasitologia , Feminino , Glicosilfosfatidilinositóis/metabolismo , Humanos , Imuno-Histoquímica , Placenta/parasitologia , Gravidez , Trofoblastos/enzimologia , Trofoblastos/parasitologiaRESUMO
Trypanosoma cruzi induces changes in the protein pattern of human placenta syncytiotrophoblast. Placental alkaline phosphatase (PLAP) is a glycoenzyme anchored to the membrane by a glycosyl-phosphatidylinositol molecule. PLAP activity and its presence was altered by the parasite in cultures of human placental villi and HEp2 cells with T.cruzi. The cells treated before the cultures with agents which affect PILAP or glycosyl-phosphatidylinositol (antibodies, PL-C, genistein, lithium) presented less parasitic invasion than the control ones. It was also observed a modification in the pattern of actine filaments of the host cells infected. We concluded that PLAP would participate in the process of T. cruzi invasion into placental syncitiotrophoblast cells, by a mechanism that involves hydrolysis of the glycosyl-phosphatidylinositol molecules, the activation of tyrosine kinase proteins, the increase of cytosolic calcium and the rearrangement of actine filaments of the host cells.
Assuntos
Animais , Feminino , Humanos , Gravidez , Doença de Chagas/enzimologia , Fosfatase Alcalina/metabolismo , Placenta/enzimologia , Trypanosoma cruzi/fisiologia , Análise de Variância , Técnicas de Cultura de Células , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Fosfatase Alcalina/análise , Glicosilfosfatidilinositóis/metabolismo , Imuno-Histoquímica , Biomarcadores , Placenta/parasitologia , Trofoblastos/enzimologia , Trofoblastos/parasitologia , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/parasitologiaRESUMO
Human term placental villi cultured "in vitro" were maintained with bloodstream forms of Trypanosoma cruzi during various periods of time. Two different concentrations of the parasite were employed. Controls contained no T. cruzi. The alkaline phosphatase activity was determined in placental villi by electron microscopy and its specific activity in the culture medium by biochemical methods. Results showed that the hemoflagellate produces a significant decrease in enzyme activity as shown by both ultracytochemical and specific activity studies and this activity was lower in cultures with high doses of parasites. The above results indicate that the reduction in enzyme activity coincides with the time of penetration and proliferation of T. cruzi in mammalian cells. These changes may represent an interaction between human trophoblast and T. cruzi.