Study of the role of caveolae/ Caveolin-1 in the early development of the human placenta

REPPETTI J.; SEYAHIAN A.; RECA A.; SIERRA M.; DAMIANO A. E.; MARTÍNEZ N.

Santiago de Chile

Workshop; International Postgraduate Workshop 2018; 2018

HEIDELBERG UNIVERSITY- PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE

Study of the role of caveolae/ Caveolin-1 in the early development of the human placenta It is well known that to achieve a successful pregnancy, it is necessary that many processes such as differentiation, proliferation and trophoblastic invasion among others, happen in a highly coordinated and synchronized manner. In the human, trophoblast cells differentiate to invasive extravillous trophoblast (EVT) or fuse to form the syncytiotrophoblasts. EVT cells proliferate, migrate and invade into the maternal decidua and the inner myometrium, remodeling the maternal spiral arteries by adopting an ?endovascular phenotype? and replace the endothelial lining of the arteries. In this way, EVT cells lose their organized epithelial phenotype and undergo a transition to a migratory and invasive mesenchymal phenotype [1]. It is well known that dysregulation of these finely controlled processes can lead to placental hypoperfusion, tissue injury, and trophoblast hypoxia and their consequences including fetal death, growth restriction, and preeclampsia.Caveolin-1 and caveolae are known to mediate molecular trafficking and have long been proposed to have a role in tumor cell migration and invasion [2]. Recently it has been linked to caveolae and caveolin-1 (Cav-1) the cell migration of endothelial cells, through the control of the composition and expansion of the plasma membrane, polarization of signal molecules and remodeling of the cytoskeleton [3]. Caveolae are specialized plasma membrane invaginations, rich in cholesterol and sphyngomyelin [4,5]. Caveolin-1 (Cav-1) is the main integral membrane protein of these subdomains and can bind to different receptors and activates multiple signaling networks.Furthermore, our laboratory reported a decreased expression of Cav-1 in trophoblast from preeclamptic placentas, which was associated to alterations in the lipid composition required for the formation of caveolae [6].We propose that these alterations may be also present at the beginning of the gestation during the formation of the placenta and may compromise cellular processes that critically depend on membrane structure. However, there are no reports about the role of caveolae at the early stages of placental development.Our objective is to investigate the role of caveolae present in the human trophoblast in the processes of proliferation, migration, invasion and endovascular differentiation and to evaluate their interaction with the maternal endothelium during the formation of the placenta.The study of the role of the caveolae in the different processes will be carried out by 2 strategies: A.Using a pharmacological approach with the drug methyl-β-cyclodextrin 5mM (MβCD) that generates disruption of the caveolae altering membrane cholesterol, B.Silencing the protein Cav-1 (siRNA-Cav1).Swan 71 cell line [7] will be cultured in DMEM-F12 supplemented with 10% fetal bovine serum, 5 mM L-Glutamine, 100 U/ mL penicillin, 100 μg/ mL streptomycin and 0.25 μg/ mL amphotericin B in humidified air at 37 ° C with 5% CO2. Cells grown exponentially throughout the study will be used. The expression of the Cav-1 will be studied by RT-PCR (sense 5?-TCTCTACACCGTTCCCATCC-3?and antisense 5?-CACAGACGGTGTGGACGTAG-3?), Western blot (Cav-1 primary antibody 1:1000) and immunofluorescence.The proliferation (studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay) and migration (studied by wound healing assay) of Swan 71 cells will be studied in control condition, after treatment with MβCD 5mM for 30 min at 37 °C and after the silencing of Cav-1 with siRNA-Cav-1.The invasiveness of Swan 71 cells will be examined through the invasion assay in inserts covered by Matrigel® and by gelatin zymography.Tube-like formation assay will also be performed.Finally, a co-culture of first trimester trophblastic cells (Swan 71) and endothelial cells will be performed. The integration of Swan71 cells with the endothelial cells under the different conditions will be studied: A) control condition, B) after treatment with MβCD 5mM and C) after the silencing of Cav-1 with siRNA-Cav-1.[1] DaSilva-Arnolda S, James JL, Al-Khan A, et al. 2015 Differentiation of first trimester cytotrophoblast to extravillous trophoblast involves an epithelialemesenchymal transition. Placenta 36 1412?1418.[2] Diaz-Valdivia N, Bravo D, Huerta H, et al. 2015 Enhanced caveolin-1 expression increases migration, anchorage-independent growth and invasion of endometrial adenocarcinoma cells. BMC Cancer 15 463.[3] Okamoto T, Schlegel A, Scherer PE & Lisanti MP 1998 Caveolins, a family of scaffolding proteins for organizing ??preassembled signaling complexes?? at the plasma membrane. The Journal of Biological Chemistry 273 5419?5422[4] Anderson RG 1998. The caveolae membrane system. Annual Review of Biochemistry 67 199?225. [5] Stan RV 2005 Structure of caveolae. Biochimica et Biophysica Acta 1746 334?348, [6] Levi L, Castro-Parodi M, Martínez N, et al 2016 The unfavorable lipid environment reduced caveolin-1 expression in apical membranes from human preeclamptic placentas. Biochimica et Biophysica Acta 1858 2171-2180 [7] Straszewski-Chavez SL, Abrahams VM, Alvero AB, et al. 2009. The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71. Placenta 30 939?948